Nematode phosphoethanolamine N-methyltransferase-like sequences

ABSTRACT

Nucleic acid molecules from nematodes encoding phosphoethanolamine n-methyltransferase polypeptides are described. PEAMT-like polypeptide sequences are also provided, as are vectors, host cells, and recombinant methods for production of PEAMT-like nucleotides and polypeptides. Also described are screening methods for identifying inhibitors and/or activators, as well as methods for antibody production.

CLAIM OF PRIORITY

This application is a divisional of U.S. application Ser. No.10/602,268, filed on Jun. 23, 2003, now U.S. Pat. No. 7,442,533, whichclaims priority to U.S. Provisional Patent Application Ser. No.60/390,490, filed on Jun. 21, 2002, all of which are hereby incorporatedby reference.

BACKGROUND

Nematodes (derived from the Greek word for thread) are active, flexible,elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved to be very successful parasitesof both plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control. CAB International, NewYork).

Nematode parasites of plants can inhabit all parts of plants, includingroots, developing flower buds, leaves, and stems. Plant parasites areclassified on the basis of their feeding habits into the broadcategories: migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

There are a very small array of chemicals available to control nematodes(Becker (1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat.Nos. 6,048,714). Nevertheless, the application of chemical nematicidesremains the major means of nematode control. In general, chemicalnematicides are highly toxic compounds known to cause substantialenvironmental damage and are increasingly restricted in the amounts andlocations in which then can be used. For example, the soil fumigantmethyl bromide which has been used effectively to reduce nematodeinfestations in a variety of specialty crops, is regulated under theU.N. Montreal Protocol as an ozone-depleting substance and is scheduledfor elimination in 2005 in the US (Carter (2001) California Agriculture,55(3):2). It is expected that strawberry and other commodity cropindustries will be significantly impacted if a suitable replacement formethyl bromide is not found. Similarly, broad-spectrum nematicides suchas Telone (various formulations of 1,3-dichloropropene) have significantrestrictions on their use because of toxicological concerns (Carter(2001) California Agriculture, Vol. 55(3):12-18).

The macrocyclic lactones (e.g., avermectins and milbemycins) anddelta-toxins from Bacillus thuringiensis (Bt) are chemicals that inprinciple provide excellent specificity and efficacy and should allowenvironmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two nematicidal agents have provenless effective in agricultural applications against root pathogens.Although certain avermectins show exquisite activity against plantparasitic nematodes these chemicals are hampered by poor bioavailabilitydue to their light sensitivity, degradation by soil microorganisms andtight binding to soil particles (Lasota & Dybas (1990) Acta Leiden59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds R. N. Perry & D. J. Wright), CAB International 1998).Consequently despite years of research and extensive use against animalparasitic nematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avermectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

Bt delta toxins must be ingested to affect their target organ, the brushborder of midgut epithelial cells (Marroquin et al. (2000) Genetics.155(4):1693-1699). Consequently they are not anticipated to be effectiveagainst the dispersal, non-feeding, juvenile stages of plant parasiticnematodes in the field. Because juvenile stages only commence feedingwhen a susceptible host has been infected, nematicides may need topenetrate the plant cuticle to be effective. Transcuticular uptake of a65-130 kDa protein—the size of typical Bt delta toxins—is unlikely.Furthermore, soil mobility is expected to be relatively poor. Eventransgenic approaches are hampered by the size of Bt delta toxinsbecause delivery in planta is likely to be constrained by the exclusionof large particles by the feeding tubes of certain plant parasiticnematodes such as Heterodera (Atkinson et al. (1998) Engineeringresistance to plant-parasitic nematodes. In: The Physiology andBiochemistry of Free-Living and Plant-parasitic Nematodes (eds R. N.Perry & D. J. Wright), CAB International 1998).

Fatty acids are a class of natural compounds that have been investigatedas alternatives to the toxic, non-specific organophosphate, carbamateand fumigant pesticides (Stadler et al. (1994) Planta Medica60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897;5,698,592; 6,124,359). It has been suggested that fatty acids derivetheir pesticidal effects by adversely interfering with the nematodecuticle or hypodermis via a detergent (solubilization) effect, orthrough direct interaction of the fatty acids and the lipophilic regionsof target plasma membranes (Davis et al. (1997) Journal of Nematology29(4S):677-684). In view of this predicted mode of action it is notsurprising that fatty acids are used in a variety of pesticidalapplications including as herbicides (e.g., SCYTHE by Dow Agrosciencesis the C9 saturated fatty acid pelargonic acid), bactericides andfungicides (U.S. Pat. Nos. 4,771,571; 5,246,716) and insecticides (e.g.,SAFER INSECTICIDAL SOAP by Safer, Inc.).

The phytotoxicity of fatty acids has been a major constraint on theirgeneral use in post-plant agricultural applications (U.S. Pat. No.5,093,124) and the mitigation of these undesirable effects whilepreserving pesticidal activity is a major area of research. Post-plantapplications are desirable because of the relatively short half-life offatty acids under field conditions.

The esterification of fatty acids can significantly decrease theirphytotoxicity (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359). Suchmodifications can however lead to loss of nematicidal activity as isseen for linoleic, linolenic and oleic acid (Stadler et al. (1994)Planta Medica 60(2):128-132) and it may be impossible to completelydecouple the phytotoxicity and nematicidal activity of pesticidal fattyacids because of their non-specific mode of action. Perhaps notsurprisingly, the nematicidal fatty acid pelargonic acid methyl ester(U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) shows a relativelysmall “therapeutic window” between the onset of pesticidal activity andthe observation of significant phytotoxicity (Davis et al. (1997) JNematol 29(4S):677-684). This is the expected result if both thephytotoxicity and the nematicidial activity derive from the non-specificdisruption of plasma membrane integrity.

Ricinoleic acid, the major component of castor oil, has been shown tohave an inhibitory effect on water and electrolyte absorption usingeverted hamster jejunal and ileal segments (Gaginella et al. (1975) JPharmacol Exp Ther 195(2):355-61) and to be cytotoxic to isolatedintestinal epithelial cells (Gaginella et al. (1977) J Pharmacol ExpTher 201(1):259-66). These features are likely the source of thelaxative properties of castor oil which is given as a purgative inhumans and livestock (e.g., castor oil is a component of some de-wormingprotocols because of its laxative properties). In contrast, the methylester of ricinoleic acid is ineffective at suppressing water absorptionin the hamster model (Gaginella et al. (1975) J Pharmacol Exp Ther195(2):355-61).

Many plant species are known to be highly resistant to nematodes. Thebest documented of these include marigolds (Tagetes spp.), rattlebox(Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castorbean (Ricinus communis), margosa (Azardiracta indica), and many membersof the family Asteraceae (family Compositae) (Hackney & Dickerson.(1975) J Nematol 7(1):84-90). In the case of the Asteraceae, thephotodynamic compound alpha-terthienyl has been shown to account for thestrong nematicidal activity of the roots. Castor beans are plowed underas a green manure before a seed crop is set. However, a significantdrawback of the castor plant is that the seed contains toxic compounds(such as ricin) that can kill humans, pets, and livestock and is alsohighly allergenic. In many cases however, the active principle(s) forplant nematicidal activity has not been discovered and it remainsdifficult to derive commercially successful nematicidal products fromthese resistant plants or to transfer the resistance to agronomicallyimportant crops such as soybeans and cotton.

Genetic resistance to certain nematodes is available in some commercialcultivars (e.g., soybeans), but these are restricted in number and theavailability of cultivars with both desirable agronomic features andresistance is limited. The production of nematode resistant commercialvarieties by conventional plant breeding based on genetic recombinationthrough sexual crosses is a slow process and is often further hamperedby a lack of appropriate germplasm.

There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

Nematode parasites of vertebrates (e.g., humans, livestock and companionanimals) include gut roundworms, hookworms, pinworms, whipworms, andfilarial worms. They can be transmitted in a variety of ways, includingby water contamination, skin penetration, biting insects, or byingestion of contaminated food.

In domesticated animals, nematode control or “de-worming” is essentialto the economic viability of livestock producers and is a necessary partof veterinary care of companion animals. Parasitic nematodes causemortality in animals (e.g., heartworm in dogs and cats) and morbidity asa result of the parasites' inhibiting the ability of the infected animalto absorb nutrients. The parasite-induced nutrient deficiency leads todisease and stunted growth in livestock and companion animals. Forinstance, in cattle and dairy herds, a single untreated infection withthe brown stomach worm can permanently restrict an animal's ability toconvert feed into muscle mass or milk.

Two factors contribute to the need for novel anthelmintics and vaccinesto control animal parasitic nematodes. First, some of the more prevalentspecies of parasitic nematodes of livestock are building resistance tothe anthelmintic drugs available currently, meaning that these productswill eventually lose their efficacy. These developments are notsurprising because few effective anthelmintic drugs are available andmost have been used continuously. Some parasitic species have developedresistance to most of the anthelmintics (Geents et al. (1997)Parasitology Today 13:149-151; Prichard (1994) Veterinary Parasitology54:259-268). The fact that many of the anthelmintic drugs have similarmodes of action complicates matters, as the loss of sensitivity of theparasite to one drug is often accompanied by side resistance—that is,resistance to other drugs in the same class (Sangster & Gill (1999)Parasitology Today 15(4):141-146). Secondly, there are some issues withtoxicity for the major compounds currently available.

Infections by parasitic nematode worms result in substantial humanmortality and morbidity, especially in tropical regions of Africa, Asia,and the Americas. The World Health Organization estimates 2.9 billionpeople are infected, and in some areas, 85% of the population carriesworms. While mortality is rare in proportion to infections, morbidity issubstantial and rivals diabetes and lung cancer in worldwide disabilityadjusted life year (DALY) measurements.

Examples of human parasitic nematodes include hookworms, filarial worms,and pinworms. Hookworms (1.3 billion infections) are the major cause ofanemia in millions of children, resulting in growth retardation andimpaired cognitive development. Filarial worm species invade thelymphatics, resulting in permanently swollen and deformed limbs(elephantiasis), and the eyes, causing African river blindness. Thelarge gut roundworm Ascaris lumbricoides infects more than one billionpeople worldwide and causes malnutrition and obstructive bowel disease.In developed countries, pinworms are common and often transmittedthrough children in daycare.

Even in asymptomatic parasitic infections, nematodes can still deprivethe host of valuable nutrients and increase the ability of otherorganisms to establish secondary infections. In some cases, infectionscan cause debilitating illnesses and can result in anemia, diarrhea,dehydration, loss of appetite, or death.

Despite some advances in drug availability and public healthinfrastructure and the near elimination of one tropical nematode (thewater-borne Guinea worm), most nematode diseases have remainedintractable problems. Treatment of hookworm diseases with anthelminticdrugs, for instance, has not provided adequate control in regions ofhigh incidence because rapid re-infection occurs after treatment. Infact, over the last 50 years, while nematode infection rates have fallenin the United States, Europe, and Japan, the overall number ofinfections worldwide has kept pace with the growing world population.Large scale initiatives by regional governments, the World HealthOrganization, foundations, and pharmaceutical companies are now underwayattempting to control nematode infections with currently availabletools, including three programs for control of Onchocerciasis (riverblindness) in Africa and the Americas using ivermectin and vectorcontrol; The Global Alliance to Eliminate Lymphatic Filariasis usingDEC, albendazole, and ivermectin; and the highly successful Guinea WormEradication Program. Until safe and effective vaccines are discovered toprevent parasitic nematode infections, anthelmintic drugs will continueto be used to control and treat nematode parasitic infections in bothhumans and domestic animals.

Finding effective compounds and vaccines against parasitic nematodes hasbeen complicated by the fact that the parasites have not been amenableto culturing in the laboratory. Parasitic nematodes are often obligateparasites (i.e., they can only survive in their respective hosts, suchas in plants, animals, and/or humans) with slow generation times. Thus,they are difficult to grow under artificial conditions, making geneticand molecular experimentation difficult or impossible. To circumventthese limitations, scientists have used Caenorhabidits elegans as amodel system for parasitic nematode discovery efforts.

C. elegans is a small free-living bacteriovorous nematode that for manyyears has served as an important model system for multicellular animals(Burglin (1998) Int. J. Parasitol. 28(3):395-411). The genome of C.elegans has been completely sequenced and the nematode shares manygeneral developmental and basic cellular processes with vertebrates(Ruvkin et al. (1998) Science 282:2033-41). This, together with itsshort generation time and ease of culturing, has made it a model systemof choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med.32:23-30).

Although C. elegans serves as a good model system for vertebrates, it isan even better model for study of parasitic nematodes, as C. elegans andother nematodes share unique biological processes not found invertebrates. For example, unlike vertebrates, nematodes produce and usechitin, have gap junctions comprised of innexin rather than connexin andcontain glutamate-gated chloride channels rather than glycine-gatedchloride channels (Bargmann (1998) Science 282:2028-33). The latterproperty is of particular relevance given that the avermectin class ofdrugs is thought to act at glutamate-gated chloride receptors and ishighly selective for invertebrates (Martin (1997) Vet. J. 154:11-34).

A subset of the genes involved in nematode-specific processes will beconserved in nematodes and absent or significantly diverged fromhomologues in other phyla. In other words, it is expected that at leastsome of the genes associated with functions unique to nematodes willhave restricted phylogenetic distributions. The completion of the C.elegans genome project and the growing database of expressed sequencetags (ESTs) from numerous nematodes facilitate identification of these“nematode-specific” genes. In addition, conserved genes involved innematode-specific processes are expected to retain the same or verysimilar functions in different nematodes. This functional equivalencehas been demonstrated in some cases by transforming C. elegans withhomologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol.246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131).This sort of functional conservations has also been shown in cross phylacomparisons for conserved genes and is expected to be more robust amongspecies within a phylum. Consequently, C. elegans and other free-livingnematode species are likely excellent surrogates for parasitic nematodeswith respect to conserved nematode processes.

Many expressed genes in C. elegans and certain genes in otherfree-living nematodes can be “knocked out” genetically by a processreferred to as RNA interference (RNAi), a technique that provides apowerful experimental tool for the study of gene function in nematodes(Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998)Proc. Natl. Acad Sci USA 95(26):15502-15507). Treatment of a nematodewith double-stranded RNA of a selected gene can destroy expressedsequences corresponding to the selected gene thus reducing expression ofthe corresponding protein. By preventing the translation of specificproteins, their functional significance and essentiality to the nematodecan be assessed. Determination of essential genes and theircorresponding proteins using C. elegans as a model system will assist inthe rational design of anti-parasitic nematode control products.

SUMMARY

The invention features nucleic acid molecules encoding Ascaris suum,Haemonchus contortus, Meloidogyne incognita, Meloidogyne javanica andStrongyloides stercoralis phosphoethanolamine n-methyltransferase-like(PEAMT-like) proteins. A. suum is the large roundworm of pigs and isclosely related to Ascaris lumbricoides, a major human pathogen. H.contortus is a parasite of ruminants (sheep, goats, cattle and otherwild ruminants) leading to emaciation, anemia and in certain casesdeath. As such it represents a major economic scourge. M. javanica andM. incognita are Root Knot Nematodes that cause substantial damage toseveral crops, including cotton, tobacco, pepper, and tomato. S.stercoralis is a nematode parasite that infects humans, primates, anddogs. It is one of the few nematodes that can multiply within its hostand can multiply unchecked in immunosuppressed individuals.

The PEAMT-like nucleic acids and polypeptides of the invention allow forthe identification of nematode species. The nucleic acids andpolypeptides of the invention also allow for the identification ofcompounds that bind to or alter the activity of PEAMT-like polypeptides.Such compounds may provide a means for combating diseases andinfestations caused by nematodes, particularly those caused by A. suumin pigs, A. lumbricoides in humans and other ascarid species in avariety of animals, H. contortus in ruminants, M. javanica and M.incognita (e.g., in tobacco, cotton, pepper, or tomato plants) and S.stercoralis (e.g., in humans, primates and dogs).

The invention is based, in part, on the identification of a cDNAencoding A. suum PEAMT1 (SEQ ID NO: 1). This 1786 nucleotide cDNA has a1380 nucleotide open reading frame (SEQ ID NO: 13) encoding a 460 aminoacid polypeptide (SEQ ID NO: 7).

The invention is also based, in part, on the identification of a cDNAencoding H. contortus PEAMT1 (SEQ ID NO: 2). This 1669 nucleotide cDNAhas a 1380 nucleotide open reading frame (SEQ ID NO: 14) encoding a 460amino acid polypeptide (SEQ ID NO: 8).

The invention is also based, in part, on the identification of a cDNAencoding M. incognita PEAMT1 (SEQ ID NO: 3). This 1472 nucleotide cDNAhas a 1371 nucleotide open reading frame (SEQ ID NO: 15) encoding a 457amino acid polypeptide (SEQ ID NO: 9).

The invention is based, in part, on the identification of a cDNAencoding S. stercoralis PEAMT1 (SEQ ID NO: 4). This 1580 nucleotide cDNAhas a 1407 nucleotide open reading frame (SEQ ID NO: 16) encoding a 469amino acid polypeptide (SEQ ID NO: 10).

The invention is also based, in part, on the identification of a cDNAencoding A. suum PEAMT2 (SEQ ID NO: 5). This 1533 nucleotide cDNA has a1311 nucleotide open reading frame (SEQ ID NO: 17) encoding a 437 aminoacid polypeptide (SEQ ID NO: 11).

The invention is based, in part, on the identification of a cDNAencoding M. javanica PEAMT2 (SEQ ID NO: 6). This 1534 nucleotide cDNAhas a 1416 nucleotide open reading frame (SEQ ID NO: 18) encoding a 472amino acid polypeptide (SEQ ID NO: 12).

In one aspect, the invention features novel nematode phosphoethanolaminen-methyltransferase-like polypeptides. Such polypeptides includepurified polypeptides having the amino acid sequences set forth in SEQID NO: 7, 8, 9, 10, 11 or 12. Also included are polypeptides having anamino acid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%,95%, or 98% identical to SEQ ID NO: 7, 8, 9, 10, 11 and/or 12 as well aspolypeptides having a sequence that differs from that of SEQ ID NO: 7,8, 9, 10, 11 or 12 at 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residues(amino acids). The purified polypeptides can be encoded by a nematodegene, e.g., a nematode gene other than C. elegans. For example, thepurified polypeptide has a sequence other than SEQ ID NO: 19, 20 or 21(C. elegans PEAMT1-like or PEAMT2-like proteins). The purifiedpolypeptides can further include a heterologous amino acid sequence,e.g., an amino-terminal or carboxy-terminal sequence. Also featured arepurified polypeptide fragments of the aforementioned PEAMT-likepolypeptides, e.g., a fragment of at least about 20, 30, 40, 50, 75, 85,104, 106, 113 150, 200, 250, 300, 350, 400, 450 or 470 amino acids.Non-limiting examples of such fragments include: fragments from aboutamino acid 1 to 50, 1 to 75, 1 to 89, 1 to 91, 1 to 99, 1 to 100, 1 to125, 51 to 113, 93 to 104, 99 to 113, 93 to 106, 228 to 262, 250 to 280,290 to 322, and 317 to 352 of SEQ ID NO: 7, 8, 9, 10, 11 and 12. Thepolypeptide or fragment thereof can be modified, e.g., processed,truncated, modified (e.g. by glycosylation, phosphorylation,acetylation, myristylation, prenylation, palmitoylation, amidation,addition of glycerophosphatidyl inositol), or any combination of theabove. Certain PEAMT-like polypeptides comprise a sequence of 400, 425,450, 475, 500 amino acids or fewer.

The invention also features polypeptides comprising, consistingessentially of or consisting of such polypeptides.

In another aspect, the invention features novel isolated nucleic acidmolecules encoding nematode PEAMT-like polypeptides. Such isolatednucleic acid molecules include nucleic acids comprising, consistingessentially of or consisting of the nucleotide sequence set forth in SEQID NO: 1, 2, 3, 4, 5 and 6 or SEQ ID NO: 13, 14, 15, 16, 17 and 18. Alsoincluded are isolated nucleic acid molecules having the same sequence asor encoding the same polypeptide as a nematode phosphoethanolaminen-methyltransferase-like gene (other than C. elegans PEAMT-like genes).

Also featured are: 1) isolated nucleic acid molecules having a strandthat hybridizes under low stringency conditions to a single strandedprobe of the sequences of SEQ ID NO: 1, 2, 3, 4, 5 and/or 6, or theircomplements and, optionally, encodes polypeptides of between 400 and 500amino acids; 2) isolated nucleic acid molecules having a strand thathybridizes under high stringency conditions to a single stranded probeof the sequence of SEQ ID NO: 1, 2, 3, 4, 5 and/or 6 or theircomplements and, optionally, encodes polypeptides of between 400 and 500amino acids; 3) isolated nucleic acid fragments of a PEAMT-like nucleicacid molecule, e.g., a fragment of SEQ ID NO: 1, 2, 3, 4, 5 and/or 6that is about 500, 750, 1000, 1250, 1500, 1750 or more nucleotides inlength or ranges between such lengths; and 4) oligonucleotides that arecomplementary to a PEAMT-like nucleic acid molecule or a PEAMT-likenucleic acid complement, e.g., an oligonucleotide of about 10, 15, 18,20, 22, 24, 28, 30, 35, 40, 50, 60, 70, 80, or more nucleotides inlength. Exemplary oligonucleotides are oligonucleotides which anneal toa site located between nucleotides about 1 to 24, 1 to 48, 1 to 60, 1 to120, 24 to 48, 24 to 60, 49 to 60, 61 to 180, 381 to 420, 421 to 480,451 to 466, 451 to 489, 451 to 516, 500 to 1450 of SEQ ID NO: 1, 2, 3,4, 5 and/or 6. Nucleic acid fragments include the following non-limitingexamples: nucleotides about 1 to 200, 100 to 300, 200 to 400, 300 to500, 300 to 466, 300 to 516, 300 to 489, 489 to 1450 of SEQ ID NO: 1, 2,3, 4, 5 and/or 6. Also within the invention are nucleic acid moleculesthat hybridize under stringent conditions to nucleic acid moleculeconsisting of SEQ ID NO: 1, 2, 3, 4, 5 or 6 and comprise 3,000, 2,000,1,000 or fewer nucleotides. The isolated nucleic acid can furtherinclude a heterologous promoter or other sequences required fortranscription or translation of the nucleic acid molecule in a cell,e.g., a mammalian or eukaryotic or prokaryotic cell, operably linked tothe PEAMT-like nucleic acid molecule. The isolated nucleic acid moleculecan encode a polypeptide having PEAMT enzymatic activity. Thus, asexplained in greater detail below, a polypeptide having PEAMT1 enzymaticactivity can catalyze the conversion of ethanolamine tomonomethylethanolamine, and a polypeptide having PEAMT2 enzymaticcatalyzes the converstion of monomethylethanolamine todimethylethanolamine and the conversion of dimethylethanolamine tocholine

A molecule featured herein can be from a nematode of the classAraeolaimida, Ascaridida, Chromadorida, Desmodorida, Diplogasterida,Monhysterida, Mononchida, Oxyurida, Rhigonematida, Spirurida, Enoplia,Desmoscolecidae, Rhabditida, or Tylenchida. Alternatively, the moleculecan be from a species of the class Rhabditida, particularly a speciesother than C. elegans or C. briggsae.

In another aspect, the invention features a vector, e.g., a vectorcontaining an aforementioned nucleic acid. The vector can furtherinclude one or more regulatory elements, e.g., a heterologous promoteror elements required for translation. The regulatory elements can beoperably linked to the phosphoethanolamine n-methyltransferase-likenucleic acid molecules in order to express a PEAMT-like nucleic acidmolecule. In yet another aspect, the invention features a transgeniccell or transgenic organism having in its genome a transgene containingan aforementioned PEAMT-like nucleic acid molecule and a heterologousnucleic acid, e.g., a heterologous promoter.

In still another aspect, the invention features an antibody, e.g., anantibody, antibody fragment, or derivative thereof that bindsspecifically to an aforementioned polypeptide. Such antibodies can bepolyclonal or monoclonal antibodies. The antibodies can be modified,e.g., humanized, rearranged as a single-chain, or CDR-grafted. Theantibodies may be directed against a fragment, a peptide, or adiscontinuous epitope from a PEAMT-like polypeptide.

In another aspect, the invention features a method of screening for acompound that binds to a nematode PEAMT-like polypeptide, e.g., anaforementioned polypeptide. The method includes providing the nematodepolypeptide; contacting a test compound to the polypeptide; anddetecting binding of the test compound to the nematode polypeptide. Inone embodiment, the method further includes contacting the test compoundto a mammalian PEAMT-like polypeptide and detecting binding of the testcompound to the mammalian PEAMT-like polypeptide. A test compound thatbinds the nematode PEAMT-like polypeptide with at least 2-fold, 5-fold,10-fold, 20-fold, 50-fold, or 100-fold affinity greater relative to itsaffinity for the mammalian (e.g., a human) PEAMT-like polypeptide can beidentified.

The invention also features methods for identifying compounds that alter(increase or decrease) the activity of a nematode phosphoethanolaminen-methyltransferase-like polypeptide. The method includes contacting thetest compound to the nematode PEAMT-like polypeptide and detecting aPEAMT-like activity. A decrease in the level of PEAMT-like activity ofthe polypeptide relative to the level of PEAMT-like activity of thepolypeptide in the absence of the test compound is an indication thatthe test compound is an inhibitor of the PEAMT-like activity. In stillanother embodiment, the method further includes contacting a testcompound such as an allosteric inhibitor or other types of inhibitorsthat prevent binding of the PEAMT-like polypeptide to other molecules orproteins. Such inhibitory compounds are potential selective agents forreducing the viability of a nematode expressing a PEAMT-likepolypeptide, e.g., A. suum, H. contortus, M. incognita M. javanicaand/or S. stercoralis. These methods can also include contacting thecompound with a plant (e.g., a spinach) phosphoethanolaminen-methyltransferase polypeptide; and detecting a PEAMT-like activity. Acompound that decreases nematode phosphoethanolamine n-methyltransferaseactivity to a greater extent than it decreases plant PEAMT-likepolypeptide activity could be useful as a selective inhibitor of thenematode polypeptide. A desirable compound can exhibit 2-fold, 5-fold,10-fold, 20-fold, 50-fold, 100-fold or greater selective activityagainst the nematode polypeptide.

Another featured method is a method of screening for a compound thatalters (increases or decreases) an activity of a phosphoethanolaminen-methyltransferase-like polypeptide or alters binding or regulation ofother polypeptides by PEAMT. The method includes providing thepolypeptide; contacting a test compound to the polypeptide; anddetecting an PEAMT-like activity or the activity of polypeptides boundor regulated by the PEAMT, wherein a change in activity of PEAMT-likepolypeptides or other downstream polypeptides relative to the PEAMT-likeactivity of the polypeptide or downstream polypeptides in the absence ofthe test compound is an indication that the test compound alters theactivity of the polypeptide(s). The method can further includecontacting the test compound to a plant (e.g., a spinach)phosphoethanolamine n-methyltransferase polypeptide and measuring themethyltransferase activity of the plant PEAMT polypeptide. A testcompound that alters the activity of the nematode PEAMT-like polypeptideat a given concentration and that does not substantially alter theactivity of the plant methyltransferase polypeptide or downstreampolypeptides at the given concentration can be identified. An additionalmethod includes screening for both binding to a PEAMT-like polypeptideand for an alteration in the activity of a PEAMT-like polypeptide. Yetanother featured method is a method of screening for a compound thatalters (increases or decreases) the viability or fitness of a transgeniccell or organism or nematode. The transgenic cell or organism has atransgene that expresses a phosphoethanolamine n-methyltransferase-likepolypeptide. The method includes contacting a test compound to thetransgenic cell or organism and detecting changes in the viability orfitness of the transgenic cell or organism. This alteration in viabilityor fitness can be measured relative to an otherwise identical cell ororganism that does not harbor the transgene.

Also featured is a method of screening for a compound that alters theexpression of a nematode nucleic acid encoding a phosphoethanolaminen-methyltransferase-like polypeptide, e.g. a nucleic acid encoding a A.suum, H. contortus M. incognita M. javanica and/or S. stercoralisPEAMT-like polypeptide. The method includes contacting a cell, e.g., anematode cell, with a test compound and detecting expression of anematode nucleic acid encoding a PEAMT-like polypeptide, e.g., byhybridization to a probe complementary to the nematode nucleic acidencoding a PEAMT-like polypeptide or by contacting polypeptides isolatedfrom the cell with a compound, e.g., antibody that binds a PEAMT-likepolypeptide. Compounds identified by the method are also within thescope of the invention.

In yet another aspect, the invention features a method of treating adisorder (e.g., an infection) caused by a nematode, e.g., A. suum, H.contortus, M. incognita, M. javanica and/or S. stercoralis, in asubject, e.g., a host plant or host animal. The method includesadministering to the subject an effective amount of an inhibitor of aPEAMT-like polypeptide activity or an inhibitor of expression of aPEAMT-like polypeptide. Non-limiting examples of such inhibitorsinclude: an antisense nucleic acid (or PNA) to an PEAMT-like nucleicacid, an antibody to a PEAMT-like polypeptide, or a small moleculeidentified as a PEAMT-like polypeptide inhibitor by a method describedherein.

A “purified polypeptide”, as used herein, refers to a polypeptide thathas been separated from other proteins, lipids, and nucleic acids withwhich it is naturally associated. The polypeptide can constitute atleast 10, 20, 50 70, 80 or 95% by dry weight of the purifiedpreparation.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample: (a) a DNA which is part of a naturally occurring genomic DNAmolecule but is not flanked by both of the nucleic acid sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones in a DNA library such as a cDNA or genomic DNAlibrary. Isolated nucleic acid molecules according to the presentinvention further include molecules produced synthetically, as well asany nucleic acids that have been altered chemically and/or that havemodified backbones.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”refers to the sequence of the nucleotides in the nucleic acid molecule,the two phrases can be used interchangeably.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the Blastall (BLASTP, BLASTX, TBLASTN,TBLASTX) or Bl2seq programs (version 2.x and later) of Altschul et al.(1990). J. Mol. Biol. 215:403-10. Bl2seq performs a comparison betweenthe subject sequence and a target sequence using either the BLASTN (usedto compare nucleic acid sequences) or BLASTP (used to compare amino acidsequences) algorithm. Typically, the default parameters of a BLOSUM62scoring matrix, gap existence cost of 11 and extension cost of 1, a wordsize of 3, an expect value of 10, a per residue cost of 1 and a lambdaratio of 0.85 are used when performing amino acid sequence alignments.The output file contains aligned regions of homology between the targetsequence and the subject sequence. Once aligned, a length is determinedby counting the number of consecutive nucleotides or amino acid residues(i.e., excluding gaps) from the target sequence that align with sequencefrom the subject sequence starting with any matched position and endingwith any other matched position. A matched position is any positionwhere an identical nucleotide or amino acid residue is present in boththe target and subject sequence. Gaps of one or more residues can beinserted into a target or subject sequence to maximize sequencealignments between structurally conserved domains (e.g., α-helices,β-sheets, and loops).

The percent identity over a particular length is determined by countingthe number of matched positions over that particular length, dividingthat number by the length and multiplying the resulting value by 100.For example, if (i) a 500 amino acid target sequence is compared to asubject amino acid sequence, (ii) the Bl2seq program presents 200 aminoacids from the target sequence aligned with a region of the subjectsequence where the first and last amino acids of that 200 amino acidregion are matches, and (iii) the number of matches over those 200aligned amino acids is 180, then the 500 amino acid target sequencecontains a length of 200 and a sequence identity over that length of 90%(i.e., 180÷200×100=90).

It will be appreciated that a nucleic acid or amino acid target sequencethat aligns with a subject sequence can result in many different lengthswith each length having its own percent identity. It is noted that thepercent identity value can be rounded to the nearest tenth. For example,78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15,78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It is also notedthat the length value will always be an integer.

The identification of conserved regions in a template, or subject,polypeptide can facilitate homologous polypeptide sequence analysis.Conserved regions can be identified by locating a region within theprimary amino acid sequence of a template polypeptide that is a repeatedsequence, forms some secondary structure (e.g., helices and betasheets), establishes positively or negatively charged domains, orrepresents a protein motif or domain. See, e.g., the Pfam web sitedescribing consensus sequences for a variety of protein motifs anddomains on the Internet at sanger.ac.uk/Pfam/ andgenome.wustl.edu/Pfam/. A description of the information included at thePfam database is described in Sonnhammer et al. (1998) Nucl. Acids Res.26: 320-322; Sonnhammer et al. (1997) Proteins 28:405-420; and Batemanet al. (1999) Nucl. Acids Res. 27:260-262. From the Pfam database,consensus sequences of protein motifs and domains can be aligned withthe template polypeptide sequence to determine conserved region(s).

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one or more subject polypeptides), which is partly orentirely heterologous, i.e., foreign, to the transgenic plant, animal,or cell into which it is introduced, or, is homologous to an endogenousgene of the transgenic plant, animal, or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe plant's genome in such a way as to alter the genome of the cell intowhich it is inserted (e.g., it is inserted at a location which differsfrom that of the natural gene or its insertion results in a knockout). Atransgene can include one or more transcriptional regulatory sequencesand other nucleic acid sequences, such as introns, that may be necessaryfor optimal expression of the selected nucleic acid, all operably linkedto the selected nucleic acid, and may include an enhancer sequence.

As used herein, the term “transgenic cell” refers to a cell containing atransgene.

As used herein, a “transgenic plant” is any plant in which one or more,or all, of the cells of the plant includes a transgene. The transgenecan be introduced into the cell, directly or indirectly by introductioninto a precursor of the cell, by way of deliberate genetic manipulation,such as by T-DNA mediated transfer, electroporation, or protoplasttransformation. The transgene may be integrated within a chromosome, orit may be extrachromosomally replicating DNA.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which affects expressionof the selected DNA sequence in specific cells of a tissue, such as aleaf, root, or stem.

As used herein, the terms “hybridizes under stringent conditions” and“hybridizes under high stringency conditions” refer to conditions forhybridization in 6× sodium chloride/sodium citrate (SSC) buffer at about45° C., followed by two washes in 0.2×SSC buffer, 0.1% SDS at 60° C. or65° C. As used herein, the term “hybridizes under low stringencyconditions” refers to conditions for hybridization in 6×SSC buffer atabout 45° C., followed by two washes in 6×SSC buffer, 0.1% (w/v) SDS at50° C.

A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

As used herein, an agent with “anthelminthic or anthelmintic orantihelminthic activity” is an agent, which when tested, has measurablenematode-killing activity or results in infertility or sterility in thenematodes such that unviable or no offspring result. In the assay, theagent is combined with nematodes, e.g., in a well of microtiter dishhaving agar media or in the soil containing the agent. Staged adultnematodes are placed on the media. The time of survival, viability ofoffspring, and/or the movement of the nematodes are measured. An agentwith “anthelminthic or anthelmintic or antihelminthic activity” reducesthe survival time of adult nematodes relative to unexposed similarlystaged adults, e.g., by about 20%, 40%, 60%, 80%, or more. In thealternative, an agent with “anthelminthic or anthelmintic orantihelminthic activity” may also cause the nematodes to ceasereplicating, regenerating, and/or producing viable progeny, e.g., byabout 20%, 40%, 60%, 80%, or more.

As used herein, the term “binding” refers to the ability of a firstcompound and a second compound that are not covalently linked tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, 0.1 nM or less.

As used herein, the term “binds specifically” refers to the ability ofan antibody to discriminate between a target ligand and a non-targetligand such that the antibody binds to the target ligand and not to thenon-target ligand when simultaneously exposed to both the given ligandand non-target ligand, and when the target ligand and the non-targetligand are both present in molar excess over the antibody.

As used herein, the term “altering an activity” refers to a change inlevel, either an increase or a decrease in the activity, (e.g., anincrease or decrease in the ability of the polypeptide to bind orregulate other polypeptides or molecules) particularly a PEAMT-like orPEAMT activity. The change can be detected in a qualitative orquantitative observation. If a quantitative observation is made, and ifa comprehensive analysis is performed over a plurality of observations,one skilled in the art can apply routine statistical analysis toidentify modulations where a level is changed and where the statisticalparameter, the p value, is less than 0.05.

In part, the nematode phosphoethanolamine n-methyltransferase proteinsand nucleic acids described herein are novel targets for anti-nematodevaccines, pesticides, and drugs. Inhibition of these molecules canprovide means of inhibiting nematode metabolism and/or the nematodelife-cycle.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts the cDNA sequence of A. summ phosphoethanolaminen-methyltransferase (PEAMT1) (SEQ ID NO: 1), its corresponding encodedamino acid sequence (SEQ ID NO: 7), and its open reading frame (SEQ IDNO: 13).

FIG. 2 depicts the cDNA sequence of H. contortus phosphoethanolaminen-methyltransferase (PEAMT1) (SEQ ID NO: 2), its corresponding encodedamino acid sequence (SEQ ID NO: 8), and its open reading frame (SEQ IDNO: 14).

FIG. 3 depicts the cDNA sequence of M. incognita phosphoethanolaminen-methyltransferase (PEAMT1) (SEQ ID NO: 3), its corresponding encodedamino acid sequence (SEQ ID NO: 9), and its open reading frame (SEQ IDNO: 15).

FIG. 4 depicts the cDNA sequence of S. stercoralis phosphoethanolaminen-methyltransferase (PEAMT1) (SEQ ID NO: 4), its corresponding encodedamino acid sequence (SEQ ID NO: 10), and its open reading frame (SEQ IDNO: 16).

FIG. 5 depicts the cDNA sequence of A. summ phosphoethanolaminen-methyltransferase (PEAMT2) (SEQ ID NO: 5), its corresponding encodedamino acid sequence (SEQ ID NO: 11), and its open reading frame (SEQ IDNO: 17).

FIG. 6 depicts the cDNA sequence of M. javanica phosphoethanolaminen-methyltransferase (PEAMT2) (SEQ ID NO: 6), its corresponding encodedamino acid sequence (SEQ ID NO: 12), and its open reading frame (SEQ IDNO: 18).

FIG. 7 is an alignment of the sequences of A. summ, H. contortus, M.incognita and S. stercoralis phosphoethanolaminen-methyltransferase-like polypeptides (SEQ ID NO: 7, 8, 9 and 10) and C.elegans PEAMT1-like polypeptides (SEQ ID NO: 19 and 20).

FIG. 8 is an alignment of the sequences of A. summ and M. javanicaphosphoethanolamine n-methyltransferase-like polypeptides (SEQ ID NO: 11and 12) and C. elegans PEAMT2-like polypeptide (SEQ ID NO: 21).

DETAILED DESCRIPTION

Choline (Cho) plays a number of important roles in biological systems.In bacteria, fungi, plants and animals, phosphatidylcholine is a majorcomponent of membrane phospholipids and the free base is a precursor tothe neurotransmitter acetylcholine in animals. Choline is also anintermediate in glycine betaine (a compound that increases tolerance toosmotic stresses) synthesis in plants (McNeil et al. (2001) Proc NatlAcad Sci USA 98:10001-5). Choline is an essential nutrient in humans andother animals, and also plays a critical role in brain development inhumans (Sheard et al. (1986) Am J Clin Nutr. 1986 43:219-24; Tayek etal. (1990) J Am Coll Nut 9:76-83). Most organisms can incorporatecholine into phosphatidylcholine using a pathway that transfers acholine moiety from CDP-choline to diacylglycerol. In similar fashion,choline precursors such as ethanolamine (EA), monomethylethanolamine(MME) and dimethylethanolamine (DME) can also be incorporated intophospholipids via the CPD-choline or Kennedy pathway. Rhizobacteria havean additional Kennedy-independent pathway that also allows theincorporation of choline excreted from plant roots directly intophospholipids (Rudder et al. (1999) J Biol Chem. 274:20011-6; Lopez-Lara& Geiger (2001) J Biotechnol 91:211-21).

Among those organisms that can synthesize choline, differentbiosynthetic pathways are used to make choline from ethanolamine via thesuccessive addition of methyl groups using S-adenosyl methionine (SAM)as the methyl donor. These pathways differ in whether they use the freebase (ethanolamine), the phosphobase (phosphoethanolamine), or thephosphatidyl base (phosphatidylethanolamine) as the methylationsubstrate. Plants are unusual in that they can methylate the free base,phosphobase or phosphatidylbase (phospholipid substrate) (Bolognese &McGraw (2000) Plant Physiol. 124(4):1800-13; Nuccio et al. (2000) J BiolChem 275(19):14095-101; Charron et al. (2002). Plant Physiol.129(1):363-73). However, the conversion of phosphatidylethanolamine tophosphatidylmonomethylethanolamine has not been demonstrated in plants,so the first methylation reaction must occur at either the free base orthe phosphobase level. It is now thought that in many plants the majorflux occurs at the phosphobase level, catalyzed by thephosphoethanolamine N-methyltransferase enzyme (PEAMT) (i.e., pEA

pMME).

In contrast, in most other organisms, methylation is carried outprimarily at the phospholipid level. The complete reaction (i.e., Ptd-EA

Ptd-MME

Ptd-DME

PtdCho) requires a single enzyme in bacteria and mammals and twoseparate enzymes in fungi (Kanipes & Henry. (1997) Biochim Biophys Acta.1348(1-2):134-41; Vance et al. (1997) Biochim Biophys Acta.1348(1-2):142-50; Hanada et al. (2001) Biosci Biotechnol Biochem.65(12):2741-8). Mammalian nerve cells are reported to have additionalphopho-base methylation activity and three distinct enzymes appear to beinvolved (Andriamampandry et al. (1992) Biochem J. 288 (1):267-72;Mukherjee et al. (1995) Neurochem Res. 20(10):1233-7).

Plant methyltransferases from spinach and Arabidopsis have been clonedby complementation of choline biosynthetic mutants in fission andbudding yeast, respectively (Bolognese & McGraw (2000) Plant Physiol.124(4):1800-13; Nuccio et al. (2000) J Biol Chem. 275(19): 14095-101).In contrast to yeast methyltransferases, which act on thephosphatidylethanolamine, these plant enzymes have been shown to act onphosphoethanolamine. A similar gene has recently been cloned fromchilled wheat tissues (Charron et al. (2002). Plant Physiol.129(1):363-73). The plant enzymes are predicted to encode solubleproteins of approximately 55 kDa that have two domains containingseparate SAM binding sites. Each domain contains motifs—termed I,post-I, II, and III—that are conserved among SAM-dependentmethyltransferases. cDNA clones encompassing partial sequence from bothSAM binding sites have been isolated from numerous plants, includingOryza sativa, Brassica napus, Gossypium hirsutum, and Hordeum vulgare.The plant methyltransferase structure is thought to have arisen from agene duplication event, since prokaryotic and animal methyltransferasesare approximately half the size of the plant enzymes and have only onemethyltransferase domain.

Some basic kinetic characteristics of the spinach methyltransferase havebeen determined from enzyme preparations isolated from fission yeastoverexpressing it. Enzyme activity is dependent on SAM andphosphoethanolamine concentrations. In the presence of these substrates,methyltransferase-containing extracts catalyze the formation ofmonomethyl- and dimethylphosphoethanolamine as well as phosphocholine.The appearance of these intermediates suggests that they are precursorsto phosphocholine. A truncated version of the spinach enzyme lacking thesecond SAM binding site can accomplish the first methylation convertingphosphoethanolamine to monomethylphosphoethanolamine, but cannot performthe second and third methylation steps. It is presumed that theC-terminal half can carry out the second and third methylationreactions.

The C. elegans genome contains two PEAMT-like genes and several homologsare found in other nematode EST datasets suggesting that these genes arewidely distributed in Nematoda. The nematode proteins and plant homologsare all presumably localized in the cytosol as in the case of the wheatPEAMT as they lack secretion leaders (analyzed by methods available onthe Internet at cbs.dtu.dk/services/TargetP) or transmembrane regions(analyzed by available on the Internet at cbs.dtu.dk/services/TNHMM).

One of the C. elegans PEAMT genes (PEAMT2) encodes a polypeptide whichis 437 amino acids long (accession number AABO4824.1, wormbase locusF54D11.1) and shows significant similarity to the C-terminal half of thespinach phosphoethanolamine n-methyltransferase and other plant homologswith two SAM binding domains. The second C. elegans PEAMT gene appearsto encode at least to two different splice variants (PEAMT1a andPEAMT1b). PEAMT1a and b are 495 and 484 amino acids long, respectively(accession number AAA81102.1, wormbase locus ZK622.3a and ZK622.3b) andare most similar to the N-terminal half of the plant PEAMTs. A PFAManalysis (available on the Internet at pfam.wustl.edu) supports theblast predictions that whereas the plant PEAMTs contain two canonicalmethyltransferase domains, the nematode proteins contain an N-terminalMT domain in PEAMT1 and a C-terminal MT domain in PEAMT2. PEAMT1 andPEAMT2 have 30-40% amino acid identity to their plant homologs in theregions that align. The similarity between PEAMT1 and PEAMT2 is low (22%amino acid identity) and is restricted to a small 127 amino acid regionin their C-terminal domains.

Given the similarity of PEAMT1 and PEAMT2 to the N- and C-terminaldomains of the plant phosphoethanolamine N-methyltransferases (e.g.spinach and Arabidopsis) respectively, their similar larval lethal RNAiphenotypes and the observation that the N-terminal half of the spinachenzyme is only capable of the first methylation reaction, we predictedthat PEAMT1 would catalyze the conversion of pEA to pMME (the firstmethylation) and PEAMT2 would catalyze the conversion of pMME to pDMEand pDME to pCHO. This hypothesis was confirmed by chemicalcomplementation of the C. elegans PEAMT1 or PEAMT2 RNAi phenotypes withEA, MME, DME or Cho (see Table 1). As predicted, the PEAMT1 larvallethal RNAi phenotype is suppressed by MME, DME and Cho but not by EAwhereas the PEAMT2 RNAi is rescued only by Cho and not by MME, DME, orEA singly or in combination.

This invention describes a novel class of nematode genes related to C.elegans proteins ZK622.3a (gi|28275069|gb|AAO38583.1|[28275069]),ZK622.3b (gi|15487647|gb|AAL00881.1|U39998_(—)4[15487647]) andF54D11.1(gi|1458245|gb|AABO4824.1|[1458245]). The nematode genes can beshown by a BLAST bioinformatics analysis and phylogenetic tree buildingto be related to the plant phosphoethanolamine n-methyltransferase genefamily. This gene family appears to be wide spread in plants andnematodes but not in arthropods, vertebrates, fungi or bacteria. We haveidentified additional homologs in the nematodes A. suum, H. contortus,M. incognita, M. javanica and S. stercoralis. Importantly, we have shownthat these proteins are essential for the viability of C. elegans usingRNAi, which together with the redundancy of choline synthesis in plantsand absence of clear homologs in vertebrates suggests that theseproteins are promising targets for anti-parasitic compounds.

The nematode proteins and plant homologs are all presumably localized inthe cytosol as in the case of the wheat PEAMT as they lack secretionleaders (analyzed by methods available on the Internet atcbs.dtu.dk/services/Target P) (http://www.cbs.dtu.dk/services/TargetP/)or transmembrane regions (analyzed by available on the Internet atcbs.dtu.dk/services.TMHMM).

The present invention provides nucleic acid sequences from nematodesencoding phosphoethanolamine n-methyltransferase-like polypeptides. TheA. suum nucleic acid molecule (SEQ ID NO: 1) and the encoded PEAMT1-likepolypeptide (SEQ ID NO: 7) are depicted in FIG. 1. The H. contortusnucleic acid molecule (SEQ ID NO: 2) and the PEAMT1-like polypeptide(SEQ ID NO: 8) are depicted in FIG. 2. The M. incognita nucleic acidmolecule (SEQ ID NO: 3) and the encoded PEAMT1-like polypeptide (SEQ IDNO: 9) are depicted in FIG. 3. The S. stercoralis nucleic acid molecule(SEQ ID NO: 4) and the PEAMT1-like polypeptide (SEQ ID NO: 10) aredepicted in FIG. 4. The A. suum nucleic acid molecule (SEQ ID NO: 5) andthe encoded PEAMT2-like polypeptide (SEQ ID NO: 11) are depicted in FIG.5. The M. javanica nucleic acid molecule (SEQ ID NO: 6) and thePEAMT2-like polypeptide (SEQ ID NO: 12) are depicted in FIG. 6. Certainsequence information for the PEAMT-like genes described herein issummarized in Table 1, below.

TABLE 1 Species CDNA ORF Polypeptide FIG. A. suum SEQ ID NO: 1 SEQ IDNO: 7 SEQ ID NO: 13 FIG. 1 H. contortus SEQ ID NO: 2 SEQ ID NO: 8 SEQ IDNO: 14 FIG. 2 M. incognita SEQ ID NO: 3 SEQ ID NO: 9 SEQ ID NO: 15 FIG.3 S. stercoralis SEQ ID NO: 4 SEQ ID NO: 10 SEQ ID NO: 16 FIG. 4 A. suumSEQ ID NO: 5 SEQ ID NO: 11 SEQ ID NO: 17 FIG. 5 M. javanica SEQ ID NO: 6SEQ ID NO: 12 SEQ ID NO: 18 FIG. 6

The invention is based, in part, on the discovery of PEAMT-likesequences from A. suum, H. contortus, M. incognita, M. javanica and S.stercoralis. The following examples are, therefore, to be construed asmerely illustrative, and not limitative of the remainder of thedisclosure in any way whatsoever. All of the publications cited hereinare hereby incorporated by reference in their entirety.

EXAMPLES

A TBLASTN query with the C. elegans genes ZK622.3a(gi|1055130|gb|AAA81102.1|[1055130]) and F54D11.1(gi|1458245|gb|AABO4824.1|[1458245]) identified multiple expressedsequence tags (ESTs are short nucleic acid fragment sequences fromsingle sequencing reads) in dbest that are predicted to encode a portionof PEAMT-like enzymes in multiple nematode species.

PEAMT1-like ESTs identified as similar to C. elegans AAA81102.1 includedAncylostoma caninum (GenBank® Identification No: 15766091), Ascaris suum(GenBank® Identification No: 17993264), Strongyloides stercoralis(GenBank® Identification No: 12714760), Haemonchus contortus (GenBank®Identification No: 27590930), multiple from Pristionchus pacificus(GenBank® Identification Nos: 6067811, 15339937, 6081336, 5816211), andMeloidogyne incognita (GenBank®Identification No: 21652426), all fromMcCarter, et al. (1999) Washington University Nematode EST Project.

PEAMT2-like ESTs identified as similar to C. elegans AAB04824.1 includedMeloidogyne javanica (GenBank® Identification No: 14624708); Meloidogyneincognita (GenBank® Identification No: 9033918); Globodera rostochiensis(GenBank® Identification No: 18080101); and multiple from Ascaris suum(GenBank® Identification Nos: 15498087, 17991691, 18688588, 17992674,18688567, 18054078, 18828817, 18688268, 18053654, 17992401, 17991763,17992578, 18689591, 18688755, 18688890, 18686360, 17993455, 17992123).

Full-Length Phosphoethanolamine n-Methyltransferase 1-Like cDNASequences

Plasmid clone, Div2728, corresponding to the S. stercoralis EST sequence(GenBank® Identification No: 12714760) was obtained from the GenomeSequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid wassequenced in its entirety. Unless otherwise indicated, all nucleotidesequences determined herein were sequenced with an automated DNAsequencer (such as model 373 from Applied Biosystems, Inc.) usingprocesses well-known to those skilled in the art. Primers used arelisted in Table 2 (see below). Full sequence data for the S. stercoralisPEAMT1 was obtained from Div2728, including nucleotide sequence forcodons 1-469 and additional 5′ and 3′ untranslated sequences.

TABLE 2 SEQ ID Name Sequence NO: Homology to T7 gtaatacgactcactatagggc22 vector polylinker primer T3 aattaaccctcactaaaggg 23 vector polylinkerprimer SP6 gatttaggtgacactatag 24 vector polylinker primer MTas-1atgcctgcggcagagcg 25 As PEAMT1 (codons 71-76) AUAP ggccacgcgtcgactagtac26 abridged universal primer SL1 gggtttaattacccaagtttga 27 nematodetranspliced leader Oligo dT ggccacgcgtcgactagtacttttttttttttttttt 28universal primer to poly A tail MU19-A atggtgaacgttcgtcgtgc 29 CePEAMT1_(a) (genomic) MU19-B catacgtatttctcatcatc 30 Ce PEAMT1_(a)(genomic) MU21-A ccagattattaccaacgccg 31 Ce PEAMT2 (genomic) MU21-Btgaacttacatagattcttg 32 Ce PEAMT2 (genomic) MTmi-9 gcaattgaatatatgcggatg33 Mi PEAMT1 (codons 192-197) MTmi-8 ctatccgaattggaatgtagcg 34 Mi PEAMT1(codons 176-181) MTmi-4 cattccaattcggatagtatc 35 Mi PEAMT1 (codons177-183) GeneRacer cgactggagcacgaggacactga 36 GeneRacer kit componentGeneRacer ne ggacactgacatggactgaaggagta 37 GeneRacer kit componentMThc-1 caacggatttcatcgaatcg 38 Hc PEAMT1 (codons 79-84) MThc-4ccacgtctttgttggttagg 39 Hc PEAMT1 (codons 50-55) RNA oligocgacuggagcacgaggacacugacauggacugaagga 40 GeneRacer kit component SL2ggttttaacccagtatctcaag 41 Haemonchus transpliced leader Met12gcatcagcaatttgatattc 42 Mj PEAMT2 (codons 302-308) Met28ccgcaatatccagaagac 43 As PEAMT2 (codons 159-164) Met39cagatctcgatacattcg 44 As PEAMT2 (codons 67-73) D2728-seqF1gttctgaaccatcaacaag 45 Ss PEAMT1 (codons 161-165) D2728-seqR1gctgaagttaatgaacatc 46 Ss PEAMT1 (codons 342-346)

Plasmid clone, Div3020, corresponding to the A. suum EST sequence(GenBank® Identification No: 17993264) was obtained from the GenomeSequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid wassequenced in its entirety. Partial sequence data for the A. suum PEAMT1was obtained from Div3020, including nucleotide sequence for codons 1-88and additional 5′ untranslated sequence. The available sequence lackedthe last 372 codons of the A. suum PEAMT1, as well as 3′ untranslatedsequence.

To obtain the missing 3′ sequence of the A. suum PEAMT1 gene, the 3′RACE technique was applied. First strand cDNA synthesis from total A.suum RNA was performed using an oligo dT primer (SEQ ID NO: 28). ThecDNA was then directly PCR amplified using a gene specific primer(MTas-1; SEQ ID NO: 25) designed from the known sequence that annealswithin the cDNA molecule of interest, and the AUAP primer, which ishomologous to the 3′ end of all cDNAs amplified with the oligo dT primer(SEQ ID NO: 28). This procedure was performed to generate clone Div3465,which contains codons 71-460 in addition to 3′ untranslated sequences.Taken together, clones Div3020 and Div3465 contain sequences comprisingthe complete open reading frame of PEAMT1 gene of A. suum.

Plasmid clone, Div3440, corresponding to the M. incognita EST sequence(GenBank® Identification No: 21652426) was obtained from the GenomeSequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid wassequenced in its entirety. Partial sequence data for the M. incognitaPEAMT1 was obtained from Div3440, including nucleotide sequence forcodons 14-227. The available sequence lacked the first 13 codons and thelast 366 codons, as well as both the 5′ and 3′ untranslated sequences.

To obtain the missing 3′ end of the M. incognita PEAMT1 gene, the 3′RACE technique was applied. First strand cDNA synthesis from total M.incognita RNA was performed using an oligo dT primer (SEQ ID NO: 28).The cDNA was then directly PCR amplified using a gene specific primer(MTmi-4; SEQ ID NO: 35) designed from the known sequence that annealswithin the cDNA molecule of interest, and the AUAP primer, which ishomologous to the 3′ end of all cDNAs amplified with the oligo dT primer(SEQ ID NO: 28). This procedure was performed to generate clone Div3640,which contains codons 177-457 in addition to 3′ untranslated sequences.

To obtain the missing 5′ sequence of the M. incognita PEAMT1 gene, the5′ oligo-capped RACE method (GeneRacer™ kit from Invitrogen LifeTechnologies) was applied. This technique results in the selectiveligation of an RNA oligonucleotide (SEQ ID NO: 40) to the 5′ ends ofdecapped mRNA using T4 RNA ligase. First strand cDNA synthesis fromtotal M. incognita oligo-capped RNA was performed using an internal genespecific primer (MTmi-9; SEQ ID NO: 33) designed from the known sequencethat anneals within the cDNA molecule of interest. The first strand cDNAwas then directly PCR amplified using a nested gene specific primer(MTmi-8; SEQ ID NO: 34) designed from known sequence that anneals withinthe cDNA molecule of interest, and the GeneRacer™ 5′ nested oligo (SEQID NO: 37), which is homologous to the 5′ end of all cDNAs amplifiedwith the GeneRacer™ oligo-capped RNA method. This procedure wasperformed to generate clone Div3845, which contains codons 1-13 inaddition to 5′ untranslated sequences. The missing nucleotide sequenceencoding codons 92-176 for the M. incognita PEAMT1 gene was obtained bythe 5′ oligo capped RACE method (GeneRacer™ kit from Invitrogen LifeTechnologies) as described previously. First strand cDNA synthesis fromtotal M. incognita oligo-capped RNA was performed using an internal genespecific primer (MTmi-9; SEQ ID NO: 33) designed from the known sequencethat anneals within the cDNA molecule of interest. The first strand cDNAwas then directly PCR amplified using a nested gene specific primer(MTmi-8; SEQ ID NO: 34) designed from known sequence that anneals withinthe cDNA molecule of interest, and the GeneRacer™5′ nested oligo (SEQ IDNO: 37), which is homologous to the 5′ end of all cDNAs amplified withthe GeneRacer™ oligo-capped RNA method. This procedure was performed togenerate clone Div3846, which contains codons 1-181. Taken together,clones Div3440, Div3845, Div3846, and Div3640 contain sequencescomprising the complete open reading frame of PEAMT1 gene of M.incognita.

Partial sequence data for the H. contortus PEAMT1 was obtained from theH. contortus EST (GenBank® Identification No: 27590930), includingnucleotide sequence for codons 3-86. The available sequence lacked thefirst 2 codons and the last 374 codons of the H. contortus PEAMT1, aswell as the 5′ and 3′ untranslated regions.

To obtain the 5′ sequence of the H. contortus PEAMT1 gene, the 5′ RACEtechnique was applied, and SL2 PCR was performed using first strand cDNAfrom H. contortus as a template (cDNA synthesis explained above). Thefirst strand cDNA was directly PCR amplified using a gene specificprimer (MThc-4; SEQ ID NO: 39) designed from known EST sequence thatanneals to a site located within the cDNA of interest, and the SL2primer (SEQ ID NO: 41), which is homologous to the 5′ end of many H.contortus cDNAs. Amplified PCR products were then cloned into a suitablevector for DNA sequence analysis. This procedure was performed to obtainclone Div3676. This clone contains codons 1-55 in addition to 5′untranslated sequences. To obtain the 3′ sequence of the H. contortusPEAMT1 gene, the 3′ RACE technique was applied. First strand cDNAsynthesis from H. contortus RNA was performed as described previously.The first strand cDNA was directly PCR amplified using a gene specificprimer (MThc-1; SEQ ID NO: 38) designed from the known sequence thatanneals within the first strand cDNA molecule of interest, and the AUAPprimer (SEQ ID NO: 26), which is homologous to the 3′ end of the cDNAmolecule of interest. This procedure was performed to generate cloneDiv3650, which contains codons 79-460 in addition to 3′ untranslatedsequences. Taken together, clones Div3650, Div3676, and the known ESTsequence contain sequences comprising the complete open reading frame ofthe PEAMT1 gene of H. contortus.

Full-Length Phosphoethanolamine n-Methyltransferase2-Like cDNA Sequences

Plasmid clone, Div2562, corresponding to the M. javanica EST sequence(GenBank® Identification No: 14624708) was obtained from the GenomeSequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid wassequenced in its entirety. Unless otherwise indicated, all nucleotidesequences determined herein were sequenced with an automated DNAsequencer (such as model 373 from Applied Biosystems, Inc.) usingprocesses well-known to those skilled in the art. Primers used forsequencing are listed in Table 2 (see below). Partial sequence data forthe M. javanica PEAMT2 was obtained from Div2562, including nucleotidesequence for codons 285-472 and additional 3′ untranslated sequence. Theclone lacked the first 284 codons of the M. javanica PEAMT2, as well asthe 5′ untranslated region.

To obtain the missing 5′ sequence of the M. javanica PEAMT2 gene, the 5′RACE technique was applied, and SL1 PCR was performed using first strandcDNA from M. javanica as a template. Briefly, SL1 PCR utilizes theobservation, that unlike most eukaryotic mRNAs, many nematode mRNAmolecules contain a common leader sequence (5′ gggtttaattacccaagtttga3′) (SEQ ID NO: 27) transpliced to their 5′ ends. If this sequence ispresent on the 5′ end of the cDNA, that cDNA can be amplified using PCRwith a primer that binds to the SL1 transpliced leader and agene-specific primer near the 3′ end of the cDNA.

Briefly, following the instructions provided by Life Technologies cDNAsynthesis kit, first strand cDNA synthesis was performed on totalnematode RNA using SuperScript™ II Reverse Transcriptase and an oligo-dTprimer (which anneals to the natural poly A tail found on the 3′ end ofall eukaryotic mRNA). RNase H was then used to degrade the original mRNAtemplate. Following degradation of the original mRNA template, the firststrand cDNA was directly PCR amplified without further purificationusing Taq DNA polymerase, a gene specific primer (Met12, SEQ ID NO: 42)designed from known sequence to the cDNA of interest, and the SL1 primer(SEQ ID NO: 27), which is homologous to the 5′ end of many nematodecDNAs. Amplified PCR products were then cloned into a suitable vectorfor DNA sequence analysis. This procedure was performed to obtain cloneDiv2474. Div2474 contains codons 1-308 in addition to 5′ untranslatedsequences. Taken together, clones Div2562 and Div2474 contain sequencescomprising the complete open reading frame of the PEAMT2 gene from M.javanica.

Partial sequence data for the A. suum PEAMT2 was obtained from the A.suum EST (GenBank® Identification No: 15498087), including nucleotidesequence for codons 35-205. The available sequence lacked the first 34codons and the last 232 codons of the A. suum PEAMT2, as well as the 5′and 3′ untranslated regions.

To obtain the 5′ sequence of the A. suum PEAMT2 gene, the 5′ RACEtechnique was applied, and SL1 PCR was performed using first strand cDNAfrom A. suum as a template (cDNA synthesis explained above). The firststrand cDNA was directly PCR amplified using a gene specific primer(Met28; SEQ ID NO: 43) designed from the known EST sequence that annealsto a site located within the cDNA of interest, and the SL1 primer (SEQID NO: 27), which is homologous to the 5′ end of many nematode cDNAs.Amplified PCR products were then cloned into a suitable vector for DNAsequence analysis. This procedure was performed to obtain clone Div2715.This clone contained codons 1-164 in addition to 5′ untranslatedsequences. To obtain the 3′ sequence of the A. suum PEAMT2 gene, the 3′RACE technique was applied. First strand cDNA synthesis from A. suum RNAwas performed as described previously. The first strand cDNA wasdirectly PCR amplified using a gene specific primer (Met39; SEQ ID NO:44) designed from the known sequence that anneals within the firststrand cDNA molecule of interest, and the AUAP primer (SEQ ID NO: 26),which is homologous to the 3′ end of the cDNA of interest. Thisprocedure was performed to generate clone Div2877, which contains codons67-437 in addition to 3′ untranslated sequences. Taken together, clonesDiv2715 and Div2877 contain sequences comprising the complete openreading frame of PEAMT2 gene of A. suum.

Characterization of Six Nematode Phosphoethanolaminen-Methyltransferases

The sequences of the four PEAMT1-like phosphoethanolaminen-methyltransferase-like nucleic acid molecules (A. suum, H. contortus,M. incognita and S. stercoralis) and two PEAMT2-like phosphoethanolaminen-methyltransferase-like nucleic acid molecules (A. suum and M.javanica) are depicted in FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5 andFIG. 6 as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5 and SEQ ID NO: 6. SEQ ID NO: 13 (A. suum) contains an open readingframe encoding a 460 amino acid polypeptide, SEQ ID NO: 14 (H.contortus) contains an open reading frame encoding a 460 amino acidpolypeptide, SEQ ID NO: 15 (M. incognita) contains an open reading frameencoding a 457 amino acid polypeptide, SEQ ID NO: 16 (S. stercoralis)contains an open reading frame encoding a 469 amino acid polypeptide,SEQ ID NO: 17 (A. suum) contains an open reading frame encoding a 437amino acid polypeptide and SEQ ID NO: 18 (M. javanica) contains an openreading frame encoding a 472 amino acid polypeptide.

The sequence of the A. suum PEAMT1-like nucleic acid molecule is recitedin FIG. 1 as SEQ ID NO: 1. This nucleotide sequence contains an openreading frame encoding a 460 amino acid polypeptide. The A. suumPEAMT1-like-protein (depicted in FIG. 1 as SEQ ID NO 7) is approximately52% identical (in the region of shared homology) to the C. elegansPEAMT1-like proteins (depicted in FIG. 7 as SEQ ID NO: 19 and 20). Thesimilarity between the PEAMT1 proteins from A. suum and from C. elegansis presented as a multiple alignment generated by the Clustal X multiplealignment program.

The sequence of the H. contortus PEAMT1-like nucleic acid molecule isrecited in FIG. 2 as SEQ ID NO: 2. This nucleotide sequence contains anopen reading frame encoding a 460 amino acid polypeptide. The H.contortus PEAMT1-like protein (depicted in FIG. 2 as SEQ ID NO 8) isapproximately 63% identical (in the region of shared homology) to the C.elegans PEAMT1-like proteins (depicted in FIG. 7 as SEQ ID NO: 19 and20). The similarity between the PEAMT1 proteins from H. contortus andfrom C. elegans is presented as a multiple alignment generated by theClustal X multiple alignment program.

The sequence of the M. incognita PEAMT1-like nucleic acid molecule isrecited in FIG. 3 as SEQ ID NO: 3. This nucleotide sequence contains anopen reading frame encoding a 457 amino acid polypeptide. The M.incognita PEAMT1-like protein (depicted in FIG. 3 as SEQ ID NO 9) isapproximately 43% identical (in the region of shared homology) to the C.elegans PEAMT1-like proteins (depicted in FIG. 7 as SEQ ID NO: 19 and20). The similarity between the PEAMT1 proteins from M. incognita andfrom C. elegans is presented as a multiple alignment generated by theClustal X multiple alignment program.

The sequence of the S. stercoralis PEAMT1-like nucleic acid molecule isrecited in FIG. 4 as SEQ ID NO: 4. This nucleotide sequence contains anopen reading frame encoding a 469 amino acid polypeptide. The S.stercoralis PEAMT1-like protein (depicted in FIG. 4 as SEQ ID NO: 10) isapproximately 36% identical (in the region of shared homology) to the C.elegans PEAMT1-like proteins (depicted in FIG. 7 as SEQ ID NO: 19 and20). The similarity between the PEAMT1 proteins from S. stercoralis andfrom C. elegans is presented as a multiple alignment generated by theClustaiX multiple alignment program.

The sequence of the A. suum PEAMT2-like nucleic acid molecule is recitedin FIG. 5 as SEQ ID NO: 5. This nucleotide sequence contains an openreading frame encoding a 437 amino acid polypeptide. The A. suumPEAMT2-like protein (depicted in FIG. 5 as SEQ ID NO 11) isapproximately 48% identical (in the region of shared homology) to the C.elegans PEAMT2-like proteins (depicted in FIG. 8 as SEQ ID NO: 21). Thesimilarity between the PEAMT2 proteins from A. suum and from C. elegansis presented as a multiple alignment generated by the Clustal X multiplealignment program.

The sequence of the M. javanica PEAMT2-like nucleic acid molecule isrecited in FIG. 6 as SEQ ID NO: 6. This nucleotide sequence contains anopen reading frame encoding a 472 amino acid polypeptide. The M.javanica PEAMT2-likeprotein (depicted in FIG. 6 as SEQ ID NO 12) isapproximately 50% identical (in the region of shared homology) to the C.elegans PEAMT2-like proteins (depicted in FIG. 8 as SEQ ID NO: 21). Thesimilarity between the PEAMT2 proteins from M. javanica and from C.elegans is presented as a multiple alignment generated by the Clustal Xmultiple alignment program.

The similarity among the A. suum, H. contortus, M. incognita, S.stercoralis, and C. elegans PEAMT1-like polypeptides is presented as amultiple alignment generated by the Clustal X multiple alignment program(depicted in FIG. 7). The similarity among the A. suum, M. javanica andC. elegans PEAMT2-like polypeptides is presented as multiple alignmentgenerated by the Clustal X multiple alignment program (depicted in FIG.8)

S-adenosylmethionine (SAM)-dependent methyltransferase proteins containfour conserved motifs which define the SAM-binding site (Kagan & Clarke(1994) Arch Biochem Biophys. 310:417-427). The four domains are referredto as motif I, post I, motif II, and motif III. The four domains arepresent in all of the PEAMT1-like proteins shown in FIG. 7 and all ofthe PEAMT2-like proteins shown in FIG. 8. Their predicted amino acidpositions in the PEAMT1-like and PEAMT2-like proteins are listed inTables 3 and 4 respectively.

TABLE 3 Amino Acid positions of conserved SAM-binding motifs in NematodePEAMT1-like proteins Nematode Motif I Post I Motif II Motif III A. suum56-63 76-80 114-120 143-152 H. contortus 56-63 76-80 114-120 143-152 M.incognita 64-71 84-88 122-128 151-160 S. stercoralis 56-63 76-80 118-124147-156 C. elegans _(a) 70-77 90-94 128-134 157-166 C. elegans _(b)79-86  99-103 137-143 166-175

TABLE 4 Amino Acid positions of conserved SAM-binding motifs in NematodePEAMT2-like proteins Nematode Motif I Post I Motif II Motif III A. suum230-238 252-256 292-298 319-328 M. javanica 254-262 276-280 316-322343-352 C. elegans 228-236 250-254 290-296 317-326

The similarity between A. suum, H. contortus, M. incognita, M. javanicaand S. stercoralis PEAMT-like sequences and other sequences were alsoinvestigated by comparison to sequence databases using BLASTP analysisagainst nr (a non-redundant protein sequence database available atwww.ncbi.nlm.nih.gov) and TBLASTN analysis against dbest (an ESTsequence database available at www.ncbi.nlm.nih.gov; top 500 hits;E=1e−4). The “Expect (E) value” is the number of sequences that arepredicted to align by chance to the query sequence with a score S orgreater given the size of the database queried. This analysis was usedto determine the potential number of plant and vertebrate homologs foreach of the nematode PEAMT-like polypeptides described above. A. suum(SEQ ID NO: 1 and 5), H. contortus (SEQ ID NO:2), M. incognita (SEQ IDNO:3), M. javanica (SEQ ID NO: 6), S. stercoralis (SEQ ID NO:4) and C.elegans (SEQ ID NO:19, 20 and 21) PEAMT-like sequences had no highscoring vertebrate hits in nr or dbest having sufficient sequencesimilarity to meet the threshold E value of 1e−4 (this E valueapproximately corresponds to a threshold for removing sequences having asequence identity of less than about 25% over approximately 100 aminoacids). Accordingly, the A. suum, H. contortus, M. incognita, M.javanica and S. stercoralis PEAMT-like enzymes of this invention do notappear to share significant sequence similarity with common vertebratemethyltransferase enzymes such as the Homo sapiens(gi|13345056|gb|AAK19172.1|[13345056]) or the Rattus norvegicus(gi|310195|gb|AAA03154.1|[310195]) phosphatidylethanolaminen-methyltransferase.

On the basis of the lack of similarity to vertebrate methyltransferasesand the redundancy of choline biosynthesis in plants, the A. suum, H.contortus, M. incognita, M. javanica and S. stercoralis PEAMT-likeenzymes are useful targets of inhibitory compounds selective for somenematodes over their hosts (e.g., humans, animals, and plants).

Functional predictions were made using BLAST with the default parameterson the nr database. BLAST searches and multiple alignment constructionwith CLUSTALX demonstrated that the C. elegans genes ZK622.3a andF54D11.1 share strong homology with the plant PEAMT genes and aretherefore related to the plant PEAMT family. Reciprocal blast searchesand phylogenetic trees confirm that the nucleotide sequences from A.suum, H. contortus, M. incognita, M. javanica and S. stercoralis areorthologs of the C. elegans gene and are therefore likely PEAMTproteins. Protein localizations were predicted using TargetP andtransmembrane domains with TMHMM. The A. suum, H. contortus, M.incognita, M. javanica and S. stercoralis PEAMT polypeptides (SEQ ID NO:7, 8, 9, 10, 11 and 12, respectively) are likely localized in thecytoplasm as in the case of the wheat PEAMT homolog, as they lacksecretion or strong mitochondrial localization predictions and have nopredicted transmembrane regions.

RNA Mediated Interference (RNAi)

A double stranded RNA (dsRNA) molecule can be used to inactivate aphosphoethanolamine N-methyl transferase (PEAMT) gene in a cell by aprocess known as RNA mediated-interference (Fire et al. (1998) Nature391:806-811, and Gonczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of a PEAMT nucleic acid(preferably exonic) or a fragment thereof. For example, the molecule cancomprise at least 50, at least 100, at least 200, at least 300, or atleast 500 or more contiguous nucleotides of a PEAMT-like gene. The dsRNAmolecule can be delivered to nematodes via direct injection, by soakingnematodes in aqueous solution containing concentrated dsRNA, or byraising bacteriovorous nematodes on E. coli genetically engineered toproduce the dsRNA molecule (Kamath et al. (2000) Genome Biol. 2; Tabaraet al. (1998) Science 282:430-431).

PEAMT RNAi by Feeding:

C. elegans can be grown on lawns of E. coli genetically engineered toproduce double-stranded RNA (dsRNA) designed to inhibit PEAMT1 or PEAMT2expression. Briefly, E. coli were transformed with genomic fragmentsencoding portions of the C. elegans PEAMT1 or the PEAMT2 gene.Specifically, a 960 nucleotide fragment was amplified from the PEAMT1gene using oligo-nucleotide primers containing the sequences5′-ATGGTGAACGTTCGTCGTGC-3′ SEQ ID NO:29 and 5′-CATACGTATTTCTCATCATC-3′SEQ ID NO:30 respectively, or an 854 nucleotide fragment was amplifiedfrom the PEAMT2 gene using oligo-nucleotide primers containing thesequences 5′-CCAGATTATTACCAACGCCG-3′ SEQ ID NO:31 and5′-TGAACTTACATAGATTCTTG-3′ SEQ ID NO:32 respectively. The PEAMT1 andPEAMT2 genomic fragments were cloned separately into an E. coliexpression vector between opposing T7 polymerase promoters. The clonewas then transformed into a strain of E. coli that carries anIPTG-inducible T7 polymerase. As a control, E. coli was transformed witha gene encoding the Green Fluorescent Protein (GFP). Feeding RNAi wasinitiated from C. elegans larvae at 23° C. on NGM plates containing IPTGand E. coli expressing the C. elegans PEAMT1 or PEAMT2, or GFP dsRNA. Ifthe starting worm (the P0) was an L1, or a dauer larva, the phenotype ofboth the PEAMT1 and PEAMT2 RNAi-generated mutants was complete or almostcomplete sterility. One the other hand, if the P0 animal was an L4larva, then the phenotype of both the PEAMT1 and PEAMT2 RNAi-generatedmutants was L1/L2 larval arrested development and lethality. Thesequence of the PEAMT1 and PEAMT2 genes is of sufficiently highcomplexity (i.e., unique) such that the RNAi is not likely to representcross reactivity with other genes.

C. elegans cultures grown in the presence of E. coli expressing dsRNAfrom the PEAMT1 or the PEAMT2 gene were strongly impaired indicatingthat the PEAMT genes provide essential functions in nematodes and thatdsRNA from the PEAMT-like genes is lethal when ingested by C. elegans.These results demonstrate that PEAMT's are important for the viabilityof C. elegans and suggest that they are useful targets for thedevelopment of compounds that reduce the viability of nematodes.

Chemical Rescue of the PEAMT1 and PEAMT2 RNAi-Generated Phenotype.

The experiments described below were designed to test whether thePEAMT1/PEAMT2 RNAi knockout phenotype can be rescued by providing C.elegans with the products downstream of the predicted PEAMT reactioncatalyzed by the enzymes. The free bases (EA, MME, DME and Cho) wereadded to the bacterial medium and it was assumed that these would betaken up and converted to the corresponding phosphobases by the actionsof ethanolamine/choline kinases.

C. elegans worms were fed bacteria expressing dsRNA homologous toPEAMT1, PEAMT2, actin, or GFP along with specific chemicals (EA, MME,DME or Cho). Chemicals were added to NGM plates at variousconcentrations and negative (GFP dsRNA) and positive (actin dsRNA)controls were performed for each chemical or chemical mixture at eachconcentration. Specifically, agar plates containing NGM and thechemicals specified in Table 1 (see below) were seeded with bacteriaexpressing double-stranded RNA homologous to either PEAMT1 or PEAMT2. Insome experiments a single L1 or dauer larva was placed on each plate,and the P0 and the F1 were examined for the next 5 days. In otherexperiments, a single L4 C. elegans hermaphrodite was placed on eachplate. The hermaphrodite was allowed to lay eggs for 24 hours and thephenotype of the F1 progeny was scored 48 hours after the initial24-hour egg-laying period. At the time of scoring, 4 individual F1progeny were cloned to separate plates containing the same chemical andbacteria they were grown on. The F1 and F2 progeny were examined overthe next 4-5 days for the presence of a phenotype.

TABLE 5 C. elegans PEAMT1 and PEAMT2 RNAi feeding phenotypes (startingwith C. elegans L1, dauer, or L4 larva as the P0 animal). Compoundsadded to the plate F1 phenotype P0 media PEAMT1 dsRNA PEAMT2 dsRNA L1None Sterility Sterility 10 mM DME Fertile adults Sterility Dauer NonePartial sterility Partial sterility 10 mM DME Fertile adults SterilityL4 None L1/L2 arrest/lethality L1/L2 arrest/lethality 10 mM ethanolamine(EA) L1/L2 arrest/lethality L1/L2 arrest/lethality 5 or 10 mM MMEFertile adults L1/L2 arrest/lethality 5 or 10 mM DME Fertile adultsL1/L2 arrest/lethality 5 mM choline (Cho) L1/L2 arrest/lethality L1/L2arrest/lethality 10 or 15 mM Cho Sterile adults L1/L2 arrest/lethality25 mM or 30 mM Cho Fertile adults Fertile adults 5 mM each EA, MMEFertile adults L1/L2 arrest/lethality 5 mM each EA, DME 5 mM each EA,Cho 5 mM each MME, DME 5 mM each MME, Cho 5 mM each DME, Cho 5 mM eachMME, DME, Cho

The C. elegans phosphoethanolamine N-methyltransferase proteins PEAMT1and PEAMT2 together catalyze the conversion of phosphoethanolamine tophosphocholine. The RNAi-generated mutants of PEAMT1 or PEAMT2 are bothpredicted to have decreased levels of choline which leads to sterility,or L1/L2 larval arrested development and death. Addition of 25 mMcholine rescues the larval arrest associated with both PEAMT1 and PEAMT2RNAi phenotypes. However, only the PEAMT1 mutants are rescued by theaddition of 5 mM monomethylethanolamine (MME) or 5 mMdimethylethanolamine (DME) while the PEAMT2 mutants are not (see Table5). These data are consistent with the prediction that PEAMT1 catalyzesthe first methylation while PEAMT2 catalyzes the second and thirdmethylations in the conversion of pEA to pCho:

Five mM DME rescues the sterility associated with PEAMT1 RNAi. Therescue by DME strongly suggests the sterility is due to a reduction incholine production and not due to other changes caused by the PEAMTmutations.

The data also demonstrate that when choline alone is used as therescuing chemical, 25 mM choline is required to complement the PEAMT1and PEAMT2 RNAi phenotypes. This suggests that chemicals that interferewith this pathway will not likely be counteracted by the amount ofcholine nematodes can acquire from the environment.

Nematicidal Activity of Small Molecules Structurally Similar toEthanolamine against Caenorhabditis elegans

The structures of ethanolamine-like molecules tested against C. elegansfor nematicidal activity are shown below.

TABLE 6 COMPOUND STRUCTURE 2-(diisopropylamino)ethanol (N-substituted)

2-(tert-butylamino)ethanol (N-substituted)

D-phenylalaninol (C2-substituted)

2-amino-1-phenylethanol (C1-substituted)

N-(2-hydroxyethyl)aniline (N-substituted)

One approach to the development of chemicals that interfere with thefunction of an enzyme is to identify compounds that mimic substratebinding but that cannot be acted on by the enzyme. Therefore, severalethanolamine-derived compounds were tested for the ability to kill C.elegans in culture. Compounds with substitutions at various positions onethanolamine were tested including some with substitutions on thenitrogen, the carbon adjacent to the nitrogen (C2), and on the carbonadjacent to the oxygen (C1).

A single C. elegans L4 larva (the P0 animal) was placed on a lawn of E.coli that had been spotted onto NGM plates containing variousconcentrations of the ethanolamine-like compounds. The growth anddevelopment of the P0 and its F1 progeny at 23° C. was monitored byvisual observation over several days. Four of the compounds tested[2-(diisopropylamino)ethanol, 2-(tert-butylamino)ethanol,D-phenylalaminol and N-(2-hydroxyethyl)aniline], showed nematicidalactivity against C. elegans. In addition, the phenotype of worms treatedwith the nematicidal ethanolamine-like compounds mimicked theRNAi-phenotype of PEAMT1 and PEAMT2. That is, the F1 progeny of thetreated worm did not develop beyond the L1/L2 stage and died. Treatmentof C. elegans with the C1-substituted compound 2-amino-1-phenylethanolshowed no nematicidal effect.

TABLE 7 Nematicidal activity of ethanolamine-like compounds against C.elegans. CONCEN- COMPOUND TRATION F1 PHENOTYPE2-(diisopropylamino)ethanol 10 mM L1/L2 arrest/lethality2-(tert-butylamino)ethanol 10 mM L1/L2 arrest/lethality D-phenylalaninol10 mM L1/L2 arrest/lethality 2-amino-1-phenylethanol 25 mM Wild-typedevelopment N-(2-hydroxyethyl)aniline 10 mM L1/L2 arrest/lethalityControl (no compound) Not applicable Wild-type developmentNematicidal Activity of Ethanolamine-Like Compounds against PlantParasites.

The ethanolamine-like compounds mentioned above are also nematicidalagainst the plant parasites Meloidogyne incongnita and Meloidogynejavanica. Between 25 and 50 J2 stage larvae were soaked in the compoundsfor 48 hours at the indicated concentrations. After the treatment, thelarvae were moved to an agar plate containing NGM. Worms that crawl awayfrom the application spot are scored as alive while those that remain atthe application spot are scored as dead. The three compounds that werenematicidal against C. elegans were also nematicidal against M.incognita and M. javanica

TABLE 8 CONCEN- % DEAD COMPOUND SPECIES TRATION WORMSdi-isopropylethanolamine M. incognita 2.5 mM 75 M. javanica 2.5 mM 702-(tert-butylamino)ethanol M. incognita   5 mM 50 M. javanica  15 mM 50D-phenylalaninol M. incognita  25 mM 100 Control (no compound) M.incognita not applicable 15Nematicidal Activity of Ethanolamine-Like Compounds against OtherNematodes.

The ethanolamine-like compounds mentioned above are also nematicidalagainst Acrobiloides ellesmerensis and Cephalobus sp. Assays were doneas those described for C. elegans L4 larvae. Three of the four compoundsthat were nematicidal against C. elegans were tested and were found tobe nematicidal against A. ellesmerensis and Cephalobus sp.

TABLE 9 COMPOUND SPECIES CONCENTRATION F1 PHENOTYPEdiisopropylamino)ethanol A. ellesmerensis 10 mM L1/L2 arrest/lethalityCephalobus sp. 10 mM L1/L2 arrest/lethality 2-(tert- A. ellesmerensis 10mM L1/L2 arrest/lethality butylamino)ethanol Cephalobus sp. 10 mM L1/L2arrest/lethality D-phenylalaninol A. ellesmerensis 12.5 mM   L1/L2arrest/lethality Cephalobus sp. 12.5 mM   L1/L2 arrest/lethality Control(no compound) Cephalobus sp. not applicable Wild-type

Sulfonic, phosphonic, or phosphate prodrugs based on the structures ofthe molecules discussed here will provide better activity than theparent molecules themselves. Enzymes like PEAMT1 and PEAMT2, whichinteract with phosphorylated substrates, bind more tightly to thephosphorylated forms of the substrate than to the non-phosphorylatedforms. For example, in the case of SH2 domains, phosphorylated peptidesexhibit binding four orders of magnitude greater than non-phosphorylatedpeptides (Bradshaw et al, (1999) J. Mol. Biol. 293(4):971-85).Therefore, the addition of a phosphate, or a phosphate mimic (e.g.,phosphonate, sulfonate) to the ethanolamine-like compounds will increasethe affinity for the enzyme making them more potent inhibitors of thePEAMT enzymes.

Identification of Additional Phosphoethanolaminen-Methyltransferase-Like Sequences

A skilled artisan can utilize the methods provided in the example aboveto identify additional nematode phosphoethanolaminen-methyltransferase-like sequences, e.g., PEAMT-like sequence fromnematodes other than A. suum, H. contortus, M. incognita, M. javanicaand S. stercoralis and/or C. elegans. In addition, nematode PEAMT-likesequences can be identified by a variety of methods includingcomputer-based database searches, hybridization-based methods, andfunctional complementation.

Database Identification. A nematode phosphoethanolaminen-methyltransferase-like sequence can be identified from a sequencedatabase, e.g., a protein or nucleic acid database using a sequencedisclosed herein as a query. Sequence comparison programs can be used tocompare and analyze the nucleotide or amino acid sequences. One suchsoftware package is the BLAST suite of programs from the National Centerfor Biotechnology Institute (NCBI; Altschul et al. (1997) Nucl. AcidsResearch 25:3389-3402). A phosphoethanolamine n-methyltransferase-likesequence of the invention can be used to query a sequence database, suchas nr, dbest (expressed sequence tag (EST) sequences), and htgs(high-throughput genome sequences), using a computer-based search, e.g.,FASTA, BLAST, or PSI-BLAST search. Homologous sequences in other species(e.g., plants and animals) can be detected in a PSI-BLAST search of adatabase such as nr (E value=10, H value=1e−2, using, for example, fouriterations; available at www.ncbi.nlm.nih.gov). Sequences so obtainedcan be used to construct a multiple alignment, e.g., a ClustalXalignment, and/or to build a phylogenetic tree, e.g., in ClustalX usingthe Neighbor-Joining method (Saitou et al. (1987) Mol. Biol. Evol.4:406-425) and bootstrapping (1000 replicates; Felsenstein (1985)Evolution 39:783-791). Distances may be corrected for the occurrence ofmultiple substitutions [D_(corr)=−ln(1−D−D²/5) where D is the fractionof amino acid differences between two sequences] (Kimura (1983) TheNeutral Theory of Molecular Evolution, Cambridge University Press).

The aforementioned search strategy can be used to identifyphosphoethanolamine n-methyltransferase-like sequences in nematodes ofthe following non-limiting, exemplary genera: Plant-parasitic nematodegenera: Afrina, Anguina, Aphelenchoides, Belonolaimus, Bursaphelenchus,Cacopaurus, Cactodera, Criconema, Criconemoides, Cryphodera,Ditylenchus, Dolichodorus, Dorylaimus, Globodera, Helicotylenchus,Hemicriconemoides, Hemicycliophora, Heterodera, Hirschmanniella,Hoplolaimus, Hypsoperine, Longidorus, Meloidogyne, Mesoanguina,Nacobbus, Nacobbodera, Panagrellus, Paratrichodorus, Paratylenchus,Pratylenchus, Pterotylenchus, Punctodera, Radopholus, Rhadinaphelenchus,Rotylenchulus, Rotylenchus, Scutellonema, Subanguina, Thecavermiculatus,Trichodorus, Turbatrix, Tylenchorhynchus, Tylenchulus, Xiphinema.

Animal- and human-parasitic nematode genera: Acanthocheilonema,Aelurostrongylus, Ancylostoma, Angiostrongylus, Anisakis, Ascaris,Ascarops, Bunostomum, Brugia, Capillaria, Chabertia, Cooperia,Crenosoma, Cyathostome species (Small Strongyles), Dictyocaulus,Dioctophyma, Dipetalonema, Dirofiliaria, Dracunculus, Draschia,Elaneophora, Enterobius, Filaroides, Gnathostoma, Gonylonema, Habronema,Haemonchus, Hyostrongylus, Lagochilascaris, Litomosoides, Loa,Mammomonogamus, Mansonella, Muellerius, Metastrongylid, Necator,Nematodirus, Nippostrongylus, Oesophagostomum, Ollulanus, Onchocerca,Ostertagia, Oxyspirura, Oxyuris, Parafilaria, Parascaris,Parastrongyloides, Parelaphostrongylus, Physaloptera, Physocephalus,Protostrongylus, Pseudoterranova, Setaria, Spirocerca, Stephanurus,Stephanofilaria, Strongyloides, Strongylus, Spirocerca, Syngamus,Teladorsagia, Thelazia, Toxascaris, Toxocara, Trichinella,Trichostrongylus, Trichuris, Uncinaria, and Wuchereria.

Particularly preferred nematode genera include: Plant: Anguina,Aphelenchoides, Belonolaimus, Bursaphelenchus, Ditylenchus,Dolichodorus, Globodera, Heterodera, Hoplolaimus, Longidorus,Meloidogyne, Nacobbus, Pratylenchus, Radopholus, Rotylenchus,Tylenchulus, Xiphinema.

Animal and human parasites: Ancylostoma, Ascaris, Brugia, Capillaria,Cooperia, Cyathostome species, Dictyocaulus, Dirofiliaria, Dracunculus,Enterobius, Haemonchus, Necator, Nematodirus, Oesophagostomum,Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parascaris, Strongyloides,Strongylus, Syngamus, Teladorsagia, Thelazia, Toxocara, Trichinella,Trichostrongylus, Trichuris, and Wuchereria.

Particularly preferred nematode species include: Plant parasites:Anguina tritici, Aphelenchoides fragariae, Belonolaimus longicaudatus,Bursaphelenchus xylophilus, Ditylenchus destructor, Ditylenchus dipsaciDolichodorus heterocephalous, Globodera pallida, Globoderarostochiensis, Globodera tabacum, Heterodera avenae, Heteroderacardiolata, Heterodera carotae, Heterodera cruciferae, Heteroderaglycines, Heterodera major, Heterodera schachtii, Heterodera zeae,Hoplolaimus tylenchiformis, Longidorus sylphus, Meloidogyne acrnea,Meloidogyne arenaria, Meloidogyne chitwoodi, Meloidogyne exigua,Meloidogyne graminicola, Meloidogyne hapla, Meloidogyne incognita,Meloidogyne javanica, Meloidogyne nassi, Nacobbus batatiformis,Pratylenchus brachyurus, Pratylenchus coffeae, Pratylenchus penetrans,Pratylenchus scribneri, Pratylenchus zeae, Radopholus similis,Rotylenchus reniformis, Tylenchulus semipenetrans, Xiphinema americanum.

Animal and human parasites: Ancylostoma braziliense, Ancylostomacaninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostomatubaeforme, Ascaris suum, Ascaris lumbrichoides, Brugia malayi,Capillaria bovis, Capillaria plica, Capillaria feliscati, Cooperiaoncophora, Cooperia punctata, Cyathostome species, Dictyocaulus filaria,Dictyocaulus viviparus, Dictyocaulus arnfieldi, Dirofiliaria immitis,Dracunculus insignis, Enterobius vermicularis, Haemonchus contortus,Haemonchus placei, Necator americanus, Nematodirus helvetianus,Oesophagostomum radiatum, Onchocerca volvulus, Onchocerca cervicalis,Ostertagia ostertagi, Ostertagia circumcincta, Oxyuris equi, Parascarisequorum, Strongyloides stercoralis, Strongylus vulgaris, Strongylusedentatus, Syngamus trachea, Teladorsagia circumcincta, Toxocara cati,Trichinella spiralis, Trichostrongylus axei, Trichostrongyluscolubriformis, Trichuris vulpis, Trichuris suis, Trichurs trichiura, andWuchereria bancrofti.

Further, a phosphoethanolamine n-methyltransferase-like sequence can beused to identify additional PEAMT-like sequence homologs within agenome. Multiple homologous copies of a PEAMT-like sequence can bepresent. For example, a nematode PEAMT-like sequence can be used as aseed sequence in an iterative PSI-BLAST search (default parameters,substitution matrix=Blosum62, gap open=11, gap extend=1) of a nonredundant database such as wormpep (E value=1e−2, H value=1e−4, using,for example 4 iterations) to determine the number of homologs in adatabase, e.g., in a database containing the complete genome of anorganism. A nematode PEAMT-like sequence can be present in a genomealong with 1, 2, 3, 4, 5, 6, 8, 10, or more homologs.

Hybridization Methods. A nematode phosphoethanolaminen-methyltransferase-like sequence can be identified by ahybridization-based method using a sequence provided herein as a probe.For example, a library of nematode genomic or cDNA clones can behybridized under low stringency conditions with the probe nucleic acid.Stringency conditions can be modulated to reduce background signal andincrease signal from potential positives. Clones so identified can besequenced to verify that they encode PEAMT-like sequences.

Another hybridization-based method utilizes an amplification reaction(e.g., the polymerase chain reaction (PCR)). Oligonucleotides, e.g.,degenerate oligonucleotides, are designed to hybridize to a conservedregion of a PEAMT-like sequence (e.g., a region conserved in the threenematode sequences depicted in FIG. 3). The oligonucleotides are used asprimers to amplify a PEAMT-like sequence from template nucleic acid froma nematode, e.g., a nematode other than A. suum, H. contortus, M.incognita, M. javanica and S. stercoralis and/or C. elegans. Theamplified fragment can be cloned and/or sequenced.

Complementation Methods. A nematode phosphoethanolaminen-methyltransferase-like sequence can be identified from acomplementation screen for a nucleic acid molecule that restoresPEAMT-like activity to a cell lacking a PEAMT-like activity. Routinemethods can be used to construct strains (i.e., nematode strains) thatlack specific enzymatic activities, e.g., PEAMT activity. For example, anematode strain mutated at the PEAMT gene locus can be identified byselecting for resistance to inhibitory compounds. Such a strain can betransformed with a plasmid library expressing nematode cDNAs. Strainscan be identified in which PEAMT activity is restored. For example, thePEAMT mutant strains transformed with the plasmid library can be exposedto allosteric inhibitors or other inhibitory compounds to select forstrains that have acquired sensitivity to the inhibitors and areexpressing a nematode PEAMT-like gene. The plasmid harbored by thestrain can be recovered to identify and/or characterize the insertednematode cDNA that provides PEAMT-like activity when expressed.

Full-length cDNA and Sequencing Methods. The following methods can beused, e.g., alone or in combination with another method describedherein, to obtain full-length nematode PEAMT-like genes and determinetheir sequences.

Plant parasitic nematodes are maintained on greenhouse pot culturesdepending on nematode preference. Root Knot Nematodes (Meloidogyne sp)are propagated on Rutgers tomato (Burpee), while Soybean Cyst Nematodes(Heterodera sp) are propagated on soybean. Total nematode RNA isisolated using the TRIZOL reagent (Gibco BRL). Briefly, 2 ml of packedworms are combined with 8 ml TRIZOL reagent and solubilized byvortexing. Following 5 minutes of incubation at room temperature, thesamples are divided into smaller volumes and spun at 14,000×g for 10minutes at 4° C. to remove insoluble material. The liquid phase isextracted with 200 μl of chloroform, and the upper aqueous phase isremoved to a fresh tube. The RNA is precipitated by the addition of 500μl of isopropanol and centrifuged to pellet. The aqueous phase iscarefully removed, and the pellet is washed in 75% ethanol and spun tore-collect the RNA pellet. The supernatant is carefully removed, and thepellet is air dried for 10 minutes. The RNA pellet is resuspended in 50μl of DEPC-H₂O and analyzed by spectrophotometry at λ 260 and 280 nm todetermine yield and purity. Yields can be 1-4 mg of total RNA from 2 mlof packed worms.

Full-length cDNAs can be generated using 5′ and 3′ RACE techniques incombination with EST sequence information. The molecular technique 5′RACE (Life Technologies, Inc., Rockville, Md.) can be employed to obtaincomplete or near-complete 5′ ends of cDNA sequences for nematodePEAMT-like cDNA sequences. Briefly, following the instructions providedby Life Technologies, first strand cDNA is synthesized from totalnematode RNA using Murine Leukemia Virus Reverse Transcriptase (M-MLVRT) and a gene specific “antisense” primer, e.g., designed fromavailable EST sequence. RNase H is used to degrade the original mRNAtemplate. The first strand cDNA is separated from unincorporated dNTPs,primers, and proteins using a GlassMAX Spin Cartridge. Terminaldeoxynucleotidyl transferase (TdT) is used to generate a homopolymericdC tailed extension by the sequential addition of dCTP nucleotides tothe 3′ end of the first strand cDNA. Following addition of the dChomopolymeric extension, the first strand cDNA is directly amplifiedwithout further purification using Taq DNA polymerase, a gene specific“antisense” primer designed from available EST sequences to anneal to asite located within the first strand cDNA molecule, and adeoxyinosine-containing primer that anneals to the homopolymeric dCtailed region of the cDNA in a polymerase chain reaction (PCR). 5′ RACEPCR amplification products are cloned into a suitable vector for furtheranalysis and sequencing.

The molecular technique, 3′ RACE (Life Technologies, Inc., Rockville,Md.), can be employed to obtain complete or near-complete 3′ ends ofcDNA sequences for nematode PEAMT-like cDNA sequences. Briefly,following the instructions provided by Life Technologies (Rockville,Md.), first strand cDNA synthesis is performed on total nematode RNAusing SuperScript™ Reverse Transcriptase and an oligo-dT primer thatanneals to the polyA tail. Following degradation of the original mRNAtemplate with RNase H, the first strand cDNA is directly PCR amplifiedwithout further purification using Taq DNA polymerase, a gene specificprimer designed from available EST sequences to anneal to a site locatedwithin the first strand cDNA molecule, and a “universal” primer whichcontains sequence identity to 5′ end of the oligo-dT primer. 3′ RACE PCRamplification products are cloned into a suitable vector for furtheranalysis and sequencing.

Nucleic Acid Variants

Isolated nucleic acid molecules of the present invention include nucleicacid molecules that have an open reading frame encoding a PEAMT-likepolypeptide. Such nucleic acid molecules include molecules having: thesequences recited in SEQ ID NO: 1, 2, 3, 4, 5 and/or 6; and sequencescoding for the PEAMT-like proteins recited in SEQ ID NO: 13, 14, 15, 16,17 and/or 18. These nucleic acid molecules can be used, for example, ina hybridization assay to detect the presence of a A. suum, H. contortus,M. incognita, M. javanica and/or S. stercoralis nucleic acid in asample.

The present invention includes nucleic acid molecules such as thoseshown in SEQ ID NO: 1, 2, 3, 4, 5 and/or 6 that may be subjected tomutagenesis to produce single or multiple nucleotide substitutions,deletions, or insertions. Nucleotide insertional derivatives of thenematode gene of the present invention include 5′ and 3′ terminalfusions as well as intra-sequence insertions of single or multiplenucleotides. Insertional nucleotide sequence variants are those in whichone or more nucleotides are introduced into a predetermined site in thenucleotide sequence, although random insertion is also possible withsuitable screening of the resulting product. Deletion variants arecharacterized by the removal of one or more nucleotides from thesequence. Nucleotide substitution variants are those in which at leastone nucleotide in the sequence has been removed and a differentnucleotide inserted in its place. Such a substitution may be silent(e.g., synonymous), meaning that the substitution does not alter theamino acid defined by the codon. Alternatively, substitutions aredesigned to alter one amino acid for another amino acid (e.g.,non-synonymous). A non-synonymous substitution can be conservative ornon-conservative. A substitution can be such that activity, e.g., aPEAMT-like activity, is not impaired. A conservative amino acidsubstitution results in the alteration of an amino acid for a similaracting amino acid, or amino acid of like charge, polarity, orhydrophobicity, e.g., an amino acid substitution listed in Table 10below. At some positions, even conservative amino acid substitutions candisrupt the activity of the polypeptide.

TABLE 10 Conservative Amino Acid Replacements Amino acid Code Replacewith any of Alanine Ala Gly, Cys, Ser Arginine Arg Lys, His AsparagineAsn Asp, Glu, Gln, Aspartic Acid Asp Asn, Glu, Gln Cysteine Cys Met,Thr, Ser Glutamine Gln Asn, Glu, Asp Glutamic Acid Glu Asp, Asn, GlnGlycine Gly Ala Histidine His Lys, Arg Isoleucine Ile Val, Leu, MetLeucine Leu Val, Ile, Met Lysine Lys Arg, His Methionine Met Ile, Leu,Val Phenylalanine Phe Tyr, His, Trp Proline Pro Serine Ser Thr, Cys, AlaThreonine Thr Ser, Met, Val Tryptophan Trp Phe, Tyr Tyrosine Tyr Phe,His Valine Val Leu, Ile, Met

The current invention also embodies splice variants of nematodePEAMT-like sequences.

Another aspect of the present invention embodies a polypeptide-encodingnucleic acid molecule that is capable of hybridizing under conditions oflow stringency (or high stringency) to the nucleic acid molecule putforth in SEQ ID NO: 1, 2, 3, 4, 5 and/or 6, or their complements.

The nucleic acid molecules that encode for phosphoethanolaminen-methyltransferase-like polypeptides may correspond to the naturallyoccurring nucleic acid molecules or may differ by one or more nucleotidesubstitutions, deletions, and/or additions. Thus, the present inventionextends to genes and any functional mutants, derivatives, parts,fragments, naturally occurring polymorphisms, homologs or analogsthereof or non-functional molecules. Such nucleic acid molecules can beused to detect polymorphisms of PEAMT genes or PEAMT-like genes, e.g.,in other nematodes. As mentioned below, such molecules are useful asgenetic probes; primer sequences in the enzymatic or chemical synthesisof the gene; or in the generation of immunologically interactiverecombinant molecules. Using the information provided herein, such asthe nucleotide sequence SEQ ID NO: 1, 2, 3, 4, 5 and/or 6, a nucleicacid molecule encoding a PEAMT-like molecule may be obtained usingstandard cloning and a screening techniques, such as a method describedherein.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, or in the form of DNA, including, for example, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. The nucleic acids may be inthe form of RNA/DNA hybrids. Single-stranded DNA or RNA can be thecoding strand, also referred to as the sense strand, or the non-codingstrand, also known as the anti-sense strand.

One embodiment of the present invention includes a recombinant nucleicacid molecule, which includes at least one isolated nucleic acidmolecule depicted in SEQ ID NO: 1, 2, 3, 4, 5, and/or 6, inserted in avector capable of delivering and maintaining the nucleic acid moleculeinto a cell. The DNA molecule may be inserted into an autonomouslyreplicating vector (suitable vectors include, for example, pGEM3Z andpcDNA3, and derivatives thereof). The vector nucleic acid may be abacteriophage DNA such as bacteriophage lambda or M13 and derivativesthereof. The vector may be either RNA or DNA, single- ordouble-stranded, prokaryotic, eukaryotic, or viral. Vectors can includetransposons, viral vectors, episomes, (e.g., plasmids), chromosomesinserts, and artificial chromosomes (e.g. BACs or YACs). Construction ofa vector containing a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include, but are not limited to, E. coli. Suitable eukaryotichosts include yeast such as S. cerevisiae, other fungi, vertebratecells, invertebrate cells (e.g., insect cells), plant cells, humancells, human tissue cells, and whole eukaryotic organisms. (e.g., atransgenic plant or a transgenic animal). Further, the vector nucleicacid can be used to generate a virus such as vaccinia or baculovirus.

The present invention also extends to genetic constructs designed forpolypeptide expression. Generally, the genetic construct also includes,in addition to the encoding nucleic acid molecule, elements that allowexpression, such as a promoter and regulatory sequences. The expressionvectors may contain transcriptional control sequences that controltranscriptional initiation, such as promoter, enhancer, operator, andrepressor sequences. A variety of transcriptional control sequences arewell known to those in the art and may be functional in, but are notlimited to, a bacterium, yeast, plant, or animal cell. The expressionvector can also include a translation regulatory sequence (e.g., anuntranslated 5′ sequence, an untranslated 3′ sequence, a poly A additionsite, or an internal ribosome entry site), a splicing sequence orsplicing regulatory sequence, and a transcription termination sequence.The vector can be capable of autonomous replication or it can integrateinto host DNA.

In an alternative embodiment, the DNA molecule is fused to a reportergene such as β-glucuronidase gene, β-galactosidase (lacZ),chloramphenicol-acetyltransferase gene, a gene encoding greenfluorescent protein (and variants thereof), or red fluorescent proteinfirefly luciferase gene, among others. The DNA molecule can also befused to a nucleic acid encoding a polypeptide affinity tag, e.g.glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine, or the influenza HA tag. The affinity tag orreporter fusion joins the reading frames of SEQ ID NO: 1, 2, 3, 4, 5and/or 6 to the reading frame of the reporter gene encoding the affinitytag such that a translational fusion is generated. Expression of thefusion gene results in translation of a single polypeptide that includesboth a nematode PEAMT-like region and reporter protein or affinity tag.The fusion can also join a fragment of the reading frame of SEQ ID NO:1, 2, 3, 4, 5 and/or 6. The fragment can encode a functional region ofthe PEAMT-like polypeptides, a structurally intact domain, or an epitope(e.g., a peptide of about 8, 10, 20, or 30 or more amino acids). Anematode PEAMT-like nucleic acid that includes at least one of aregulatory region (e.g., a 5′ regulatory region, a promoter, anenhancer, a 5′ untranslated region, a translational start site, a 3′untranslated region, a polyadenylation site, or a 3′ regulatory region)can also be fused to a heterologous nucleic acid. For example, thepromoter of a PEAMT-like nucleic acid can be fused to a heterologousnucleic acid, e.g., a nucleic acid encoding a reporter protein.

Suitable cells to transform include any cell that can be transformedwith a nucleic acid molecule of the present invention. A transformedcell of the present invention is also herein referred to as arecombinant or transgenic cell. Suitable cells can either beuntransformed cells or cells that have already been transformed with atleast one nucleic acid molecule. Suitable cells for transformationaccording to the present invention can either be: (i) endogenouslycapable of expressing the PEAMT-like protein or; (ii) capable ofproducing such protein after transformation with at least one nucleicacid molecule of the present invention.

In an exemplary embodiment, a nucleic acid of the invention is used togenerate a transgenic nematode strain, e.g., a transgenic C. elegansstrain. To generate such a strain, nucleic acid is injected into thegonad of a nematode, thus generating a heritable extrachromosomal arraycontaining the nucleic acid (see, e.g., Mello et al. (1991) EMBO J.10:3959-3970). The transgenic nematode can be propagated to generate astrain harboring the transgene. Nematodes of the strain can be used inscreens to identify inhibitors specific for a A. suum, H. contortus, M.incognita, M. javanica and/or S. stercoralis PEAMT-like gene.

Oligonucleotides

Also provided are oligonucleotides that can form stable hybrids with anucleic acid molecule of the present invention. The oligonucleotides canbe about 10 to 200 nucleotides, about 15 to 120 nucleotides, or about 17to 80 nucleotides in length, e.g., about 10, 20, 30, 40, 50, 60, 80,100, 120 nucleotides in length. The oligonucleotides can be used asprobes to identify nucleic acid molecules, primers to produce nucleicacid molecules, or therapeutic reagents to inhibit nematode PEAMT-likeprotein activity or production (e.g., antisense, triplex formation,ribozyme, and/or RNA drug-based reagents). The present inventionincludes oligonucleotides of RNA (ssRNA and dsRNA), DNA, or derivativesof either. The invention extends to the use of such oligonucleotides toprotect non-nematode organisms (for example e.g., plants and animals)from disease by reading the viability of infecting namatodes, e.g.,using a technology described herein. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto a non-nematode organism using techniques known to those skilled inthe art, including, but not limited to, transgenic expression in plantsor animals.

Primer sequences can be used to amplify a phosphoethanolaminen-methyltransferase-like nucleic acid or fragment thereof. For example,at least 10 cycles of PCR amplification can be used to obtain such anamplified nucleic acid. Primers can be at least about 8-40, 10-30 or14-25 nucleotides in length, and can anneal to a nucleic acid “templatemolecule”, e.g., a template molecule encoding an PEAMT-like geneticsequence, or a functional part thereof, or its complementary sequence.The nucleic acid primer molecule can be any nucleotide sequence of atleast 10 nucleotides in length derived from, or contained withinsequences depicted in SEQ ID NO: 1, 2, 3, 4, 5 and/or 6 and theircomplements. The nucleic acid template molecule may be in a recombinantform, in a virus particle, bacteriophage particle, yeast cell, animalcell, plant cell, fungal cell, or bacterial cell. A primer can bechemically synthesized by routine methods.

This invention embodies any phosphoethanolamine n-methyltransferase-likesequences that are used to identify and isolate similar genes from otherorganisms, including nematodes, prokaryotic organisms, and othereukaryotic organisms, such as other animals and/or plants.

In another embodiment, the invention provides oligonucleotides that arespecific for a A. suum, H. contortus, M. incognita, M. javanica and/orS. stercoralis PEAMT-like nucleic acid molecule. Such oligonucleotidescan be used in a PCR test to determine if a A. suum, H. contortus, M.incognita, M. javanica and/or S. stercoralis nucleic acid is present ina sample, e.g., to monitor a disease caused A. suum, H. contortus, M.incognita, M. javanica and/or S. stercoralis.

Protein Production

Isolated phosphoethanolamine n-methyltransferase-like proteins fromnematodes can be produced in a number of ways, including production andrecovery of the recombinant proteins and/or chemical synthesis of theprotein. In one embodiment, an isolated nematode PEAMT-like protein isproduced by culturing a cell, e.g., a bacterial, fungal, plant, oranimal cell, capable of expressing the protein, under conditions foreffective production and recovery of the protein. The nucleic acid canbe operably linked to a heterologous promoter, e.g., an induciblepromoter or a constitutive promoter. Effective growth conditions aretypically, but not necessarily, in liquid media comprising salts, water,carbon, nitrogen, phosphate sources, minerals, and other nutrients, butmay be any solution in which PEAMT-like proteins may be produced.

In one embodiment, recovery of the protein may refer to collecting thegrowth solution and need not involve additional steps of purification.Proteins of the present invention, however, can be purified usingstandard purification techniques, such as, but not limited to, affinitychromatography, thermaprecipitation, immunoaffinity chromatography,ammonium sulfate precipitation, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, and others.

The phosphoethanolamine n-methyltransferase-like polypeptide can befused to an affinity tag, e.g., a purification handle (e.g.,glutathione-S-reductase, hexa-histidine, maltose binding protein,dihydrofolate reductases, or chitin binding protein) or an epitope tag(e.g., c-myc epitope tag, FLAG™ tag, or influenza HA tag). Affinitytagged and epitope tagged proteins can be purified using routineart-known methods.

Antibodies against Phosphoethanolamine n-Methyltransferase-LikePolypeptides

Recombinant phosphoethanolamine n-methyltransferase-like gene productsor derivatives thereof can be used to produce immunologicallyinteractive molecules, such as antibodies, or functional derivativesthereof. Useful antibodies include those that bind to a polypeptide thathas substantially the same sequence as the amino acid sequences recitedin SEQ ID NO: 7, 8, 9, 10, 11 and/or 12, or that has at least 60%similarity over 50 or more amino acids to these sequences. In apreferred embodiment, the antibody specifically binds to a polypeptidehaving the amino acid sequence recited in SEQ ID NO: 7, 8, 9, 10, 11and/or 12. The antibodies can be antibody fragments and geneticallyengineered antibodies, including single chain antibodies or chimericantibodies that can bind to more than one epitope. Such antibodies maybe polyclonal or monoclonal and may be selected from naturally occurringantibodies or may be specifically raised to a recombinant PEAMT-likeprotein.

Antibodies can be derived by immunization with a recombinant or purifiedPEAMT-like gene or gene product. As used herein, the term “antibody”refers to an immunoglobulin, or fragment thereof. Examples of antibodyfragments include F(ab) and F(ab′)₂ fragments, particularly functionalones able to bind epitopes. Such fragments can be generated byproteolytic cleavage, e.g., with pepsin, or by genetic engineering.Antibodies can be polyclonal, monoclonal, or recombinant. In addition,antibodies can be modified to be chimeric, or humanized. Further, anantibody can be coupled to a label or a toxin.

Antibodies can be generated against a full-length PEAMT-like protein, ora fragment thereof, e.g., an antigenic peptide. Such polypeptides can becoupled to an adjuvant to improve immunogenicity. Polyclonal serum isproduced by injection of the antigen into a laboratory animal such as arabbit and subsequent collection of sera. Alternatively, the antigen isused to immunize mice. Lymphocytic cells are obtained from the mice andfused with myelomas to form hybridomas producing antibodies.

Peptides for generating phosphoethanolamine n-methyltransferase-likeantibodies can be about 8, 10, 15, 20, 30 or more amino acid residues inlength, e.g., a peptide of such length obtained from SEQ ID NO: 7, 8, 9,10, 11 and/or 12. Peptides or epitopes can also be selected from regionsexposed on the surface of the protein, e.g., hydrophilic or amphipathicregions. An epitope in the vicinity of the active or binding site can beselected such that an antibody binding such an epitope would blockaccess to the active site or prevent binding. Antibodies reactive with,or specific for, any of these regions, or other regions or domainsdescribed herein are provided. An antibody to a PEAMT-like protein canmodulate a PEAMT-like activity.

Monoclonal antibodies, which can be produced by routine methods, areobtained in abundance and in homogenous form from hybridomas formed fromthe fusion of immortal cell lines (e.g., myelomas) with lymphocytesimmunized with PEAMT-like polypeptides such as those set forth in SEQ IDNO: 7, 8, 9, 10, 11 and/or 12.

In addition, antibodies can be engineered, e.g., to produce a singlechain antibody (see, for example, Colcher et al. (1999) Ann N Y Acad Sci880: 263-280; and Reiter (1996) Clin Cancer Res 2: 245-252). In stillanother implementation, antibodies are selected or modified based onscreening procedures, e.g., by screening antibodies or fragments thereoffrom a phage display library.

Antibodies of the present invention have a variety of important useswithin the scope of this invention. For example, such antibodies can beused: (i) as therapeutic compounds to passively immunize an animal inorder to protect the animal from nematodes susceptible to antibodytreatment; (ii) as reagents in experimental assays to detect presence ofnematodes; (iii) as tools to screen for expression of the gene productin nematodes, animals, fungi, bacteria, and plants; and/or (iv) as apurification tool of PEAMT-like protein; (v) as PEAMTinhibitors/activators that can be expressed or introduced into plants oranimals for therapeutic purposes.

An antibody against a phosphoethanolamine n-methyltransferase-likeprotein can be produced in a plant cell, e.g., in a transgenic plant orin culture (see, e.g., U.S. Pat. No. 6,080,560).

Antibodies that specifically recognize a A. suum, H. contortus, M.incognita, M. javanica and/or S. stercoralis PEAMT-like proteins can beused to identify A. suum, H. contortus, M. incognita, M. javanica and/orS. stercoralis nematodes, and, thus, can be used to monitor a diseasecaused by A. suum, H. contortus, M. incognita, M. javanica and/or S.stercoralis.

Nucleic Acids Agents

Also featured are isolated nucleic acids that are antisense to nucleicacids encoding nematode phosphoethanolamine n-methyltransferase-likeproteins. An “antisense” nucleic acid includes a sequence that iscomplementary to the coding strand of a nucleic acid encoding aPEAMT-like protein. The complementarity can be in a coding region of thecoding strand or in a noncoding region, e.g., a 5′ or 3′ untranslatedregion, e.g., the translation start site. The antisense nucleic acid canbe produced from a cellular promoter (e.g., a RNA polymerase II or IIIpromoter), or can be introduced into a cell, e.g., using a liposome. Forexample, the antisense nucleic acid can be a synthetic oligonucleotidehaving a length of about 10, 15, 20, 30, 40, 50, 75, 90, 120 or morenucleotides in length.

An antisense nucleic acid can be synthesized chemically or producedusing enzymatic reagents, e.g., a ligase. An antisense nucleic acid canalso incorporate modified nucleotides, and artificial backbonestructures, e.g., phosphorothioate derivative, and acridine substitutednucleotides.

Ribozymes. The antisense nucleic acid can be a ribozyme. The ribozymecan be designed to specifically cleave RNA, e.g., a PEAMT-like mRNA.Methods for designing such ribozymes are described in U.S. Pat. No.5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591. Forexample, the ribozyme can be a derivative of Tetrahymena L-19 IVS RNA inwhich the nucleotide sequence of the active site is modified to becomplementary to a PEAMT-like nucleic acid (see, e.g., Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).

Peptide Nucleic acid (PNA). An antisense agent directed against anphosphoethanolamine n-methyltransferase-like nucleic acid can be apeptide nucleic acid (PNA). See Hyrup et al. (1996) Bioorganic &Medicinal Chemistry 4: 5-23) for methods and a description of thereplacement of the deoxyribose phosphate backbone for a pseudopeptidebackbone. A PNA can specifically hybridize to DNA and RNA underconditions of low ionic strength as a result of its electrostaticproperties. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93:14670-14675.

RNA Mediated Interference (RNAi). A double stranded RNA (dsRNA) moleculecan be used to inactivate a phosphoethanolamine n-methyltransferase-likegene in a cell by a process known as RNA mediated-interference (RNAi;e.g., Fire et al. (1998) Nature 391:806-811, and Gönczy et al. (2000)Nature 408:331-336). The dsRNA molecule can have the nucleotide sequenceof a PEAMT-like nucleic acid described herein or a fragment thereof. Themolecule can be injected into a cell, or a syncytium, e.g., a nematodegonad as described in Fire et al., supra.

Screening Assays

Another embodiment of the present invention is a method of identifying acompound capable of altering (e.g., inhibiting or enhancing) theactivity of PEAMT-like molecules. This method, also referred to as a“screening assay,” herein, includes, but is not limited to, thefollowing procedure: (i) contacting an isolated PEAMT-like protein witha test inhibitory compound under conditions in which, in the absence ofthe test compound, the protein has PEAMT-like activity; and (ii)determining if the test compound alters the PEAMT-like activity.Suitable inhibitors or activators that alter a nematode PEAMT-likeactivity include compounds that interact directly with a nematodePEAMT-like protein, perhaps but not necessarily, in the active orbinding site. They can also interact with other regions of the nematodePEAMT protein by binding to regions outside of the active site or siteresponsible for regulation, for example, by allosteric interaction.

In one embodiment the A. suum, H. contortus, M. incognita, M. javanicaor S. stercoralis PEAMT is expressed in a yeast or bacterial cell andthen purified and screened in a TLC based radioactivity assay (Bolognese& McGraw (2000) Plant Physiol. 124(4):1800-13; Nuccio et al. (2000) JBiol Chem. 275(19):14095-101; Charron et al. (2002) Plant Physiol.129(1):363-73). ¹⁴C-labelled S-adenosyl-methionine (¹⁴C-SAM) co-factoris used and the conversion of phosphoethanolamine (pEA),phosphomonomethylethanolamine (pMME), or phosphodimethylethanolamine(pDME) to ¹⁴C-labelled pMME, pDME or phosphocholine (pCho) is monitoredafter TLC separation. Compounds that decrease the conversion of pEA,pMME or pDME to pMME, pDME or pCho are candidate PEAMT inhibitors.

Compounds. A test compound can be a large or small molecule, forexample, an organic compound with a molecular weight of about 100 to10,000; 200 to 5,000; 200 to 2000; or 200 to 1,000 daltons. A testcompound can be any chemical compound, for example, a small organicmolecule, a carbohydrate, a lipid, an amino acid, a polypeptide, anucleoside, a nucleic acid, or a peptide nucleic acid. Small moleculesinclude, but are not limited to, metabolites, metabolic analogues,peptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e., includingheteroorganic and organometallic compounds). Compounds and componentsfor synthesis of compounds can be obtained from a commercial chemicalsupplier, e.g., Sigma-Aldrich Corp. (St. Louis, Mo.). The test compoundor compounds can be naturally occurring, synthetic, or both. A testcompound can be the only substance assayed by the method describedherein. Alternatively, a collection of test compounds can be assayedeither consecutively or concurrently by the methods described herein.

Compounds can also act by allosteric inhibition or directly bypreventing the substrate phosphoethanolamine,phosphomonomethylethanolamine, phosphodimethylethanolamine or thecofactor S-adenosylmethionine from binding to the enzyme, and thus,regulating its target, i.e., a phosphoethanolamine n-methyltransferase.

A high-throughput method can be used to screen large libraries ofchemicals. Such libraries of candidate compounds can be generated orpurchased, e.g., from Chembridge Corp. (San Diego, Calif.). Librariescan be designed to cover a diverse range of compounds. For example, alibrary can include 10,000, 50,000, or 100,000 or more unique compounds.Merely by way of illustration, a library can be constructed fromheterocycles including pyridines, indoles, quinolines, furans,pyrimidines, triazines, pyrroles, imidazoles, naphthalenes,benzimidazoles, piperidines, pyrazoles, benzoxazoles, pyrrolidines,thiphenes, thiazoles, benzothiazoles, and morpholines. A library can bedesigned and synthesized to cover such classes of chemicals, e.g., asdescribed in DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A.90:6909-6913; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422-11426; Zuckermann et al. (1994) J. Med. Chem. 37:2678-2685; Choet al. (1993) Science 261:1303-1305; Carrell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233-1251.

Organism-based Assays. Organisms can be grown in microtiter plates,e.g., 6-well, 32-well, 64-well, 96-well, 384-well plates.

In one embodiment, the organism is a nematode. The nematodes can begenetically modified. Non-limiting examples of such modified nematodesinclude: 1) nematodes or nematode cells (A. suum, H. contortus, M.incognita, M. javanica, S. stercoralis, and/or C. elegans) having one ormore PEAMT-like genes inactivated (e.g., using RNA mediatedinterference); 2) nematodes or nematode cells expressing a heterologousPEAMT-like gene, e.g., an PEAMT-like gene from another species; and 3)nematodes or nematode cells having one or more endogenous PEAMT-likegenes inactivated and expressing a heterologous PEAMT-like gene, e.g., aA. suum, H. contortus, M. incognita, M. javanica and/or S. stercoralisPEAMT-like gene as described herein.

A plurality of candidate compounds, e.g., a combinatorial library, canbe screened. The library can be provided in a format that is amenablefor robotic manipulation, e.g., in microtitre plates. Compounds can beadded to the wells of the microtiter plates. Following compound additionand incubation, viability and/or reproductive properties of thenematodes or nematode cells are monitored.

The compounds can also be pooled, and the pools tested. Positive poolsare split for subsequent analysis. Regardless of the method, compoundsthat decrease the viability or reproductive ability of nematodes,nematode cells, or progeny of the nematodes are considered leadcompounds.

In another embodiment, the compounds can be tested on a microorganism ora eukaryotic or mammalian cell line, e.g., rabbit skin cells, Chinesehamster ovary cells (CHO), and/or Hela cells. For example, CHO cellsabsent for PEAMT-like genes, but expressing a nematode PEAMT-like genecan be used. The generation of such strains is routine in the art. Asdescribed above for nematodes and nematode cells, the cell lines can begrown in microtitre plates, each well having a different candidatecompound or pool of candidate compounds. Growth is monitored during orafter the assay to determine if the compound or pool of compounds is amodulator of a nematode PEAMT-like polypeptide.

In Vitro Activity Assays. The screening assay can be an in vitroactivity assay. For example, a nematode phosphoethanolaminen-methyltransferase-like polypeptide can be purified as described above.The polypeptide can be disposed in an assay container, e.g., a well of amicrotitre plate. A candidate compound can be added to the assaycontainer, and the PEAMT-like activity is measured. Optionally, theactivity is compared to the activity measured in a control container inwhich no candidate compound is disposed or in which an inert ornon-functional compound is disposed

In Vitro Binding Assays. The screening assay can also be a cell-freebinding assay, e.g., an assay to identify compounds that bind a nematodePEAMT-like polypeptide. For example, a nematode PEAMT-like polypeptidecan be purified and labeled. The labeled polypeptide is contacted tobeads; each bead has a tag detectable by mass spectroscopy, and testcompound, e.g., a compound synthesized by combinatorial chemicalmethods. Beads to which the labeled polypeptide is bound are identifiedand analyzed by mass spectroscopy. The beads can be generated using“split-and-pool” synthesis. The method can further include a secondassay to determine if the compound alters the activity of the PEAMT-likepolypeptide.

Optimization of a Compound. Once a lead compound has been identified,standard principles of medicinal chemistry can be used to producederivatives of the compound. Derivatives can be screened for improvedpharmacological properties, for example, efficacy, pharmacokinetics,stability, solubility, and clearance. The moieties responsible for acompound's activity in the above-described assays can be delineated byexamination of structure-activity relationships (SAR) as is commonlypracticed in the art. One can modify moieties on a lead compound andmeasure the effects of the modification on the efficacy of the compoundto thereby produce derivatives with increased potency. For an example,see Nagarajan et al. (1988) J. Antibiot. 41:1430-1438. A modificationcan include N-acylation, amination, amidation, oxidation, reduction,alkylation, esterification, and hydroxylation. Furthermore, if thebiochemical target of the lead compound is known or determined, thestructure of the target and the lead compound can inform the design andoptimization of derivatives. Molecular modeling software to do this iscommercially available (e.g., Molecular Simulations, Inc.). “SAR byNMR,” as described in Shuker et al. (1996) Science 274:1531-1534, can beused to design ligands with increased affinity, by joininglower-affinity ligands.

A preferred compound is one that interferes with the function of aphosphoethanolamine n-methyltransferase-like polypeptide and that is notsubstantially toxic to plants, animals, or humans. By “not substantiallytoxic” it is meant that the compound does not substantially affect therespective animal, or human PEAMT proteins or phosphoethanolaminen-methyltransferase activity. Thus, particularly desirable inhibitors ofA. suum, H. contortus, M. incognita, M. javanica and/or S. stercoralisPEAMT do not substantially inhibit non-nematode PEAMT-like polypeptidesor phosphatidylethanolamine n-methyltransferase activity of vertebrates,e.g., humans for example. Other desirable compounds do not substantiallyinhibit to phosphoethanolamine n-methyltransferase activity of plantssuch as tomato (GenBank® Identification No: 12584943), spinach (GenBank®Identification No: 7407189), or wheat (GenBank® Identification No:17887465).

Standard pharmaceutical procedures can be used to assess the toxicityand therapeutic efficacy of a modulator of a PEAMT-like activity. TheLD50 (the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population can be measured incell cultures, experimental plants (e.g., in laboratory or fieldstudies), or experimental animals. Optionally, a therapeutic index canbe determined which is expressed as the ratio: LD50/ED50. Hightherapeutic indices are indicative of a compound being an effectivePEAMT-like inhibitor, while not causing undue toxicity or side effectsto a subject (e.g., a host plant or host animal).

Alternatively, the ability of a candidate compound to modulate anon-nematode phosphoethanolamine n-methyltransferase-like polypeptide isassayed, e.g., by a method described herein. For example, the inhibitionconstant of a candidate compound for a mammalian PEAMT-like polypeptidecan be measured and compared to the inhibition constant for a nematodePEAMT-like polypeptide.

The aforementioned analyses can be used to identify and/or design amodulator with specificity for nematode phosphoethanolaminen-methyltransferase-like polypeptide over vertebrate or other animal(e.g., mammalian) phosphatidylethanolamine n-methyltransferase-likepolypeptides. Suitable nematodes to target are any nematodes with thePEAMT-like proteins or proteins that can be targeted by a compound thatotherwise inhibits, reduces, activates, or generally affects theactivity of nematode PEAMT proteins.

Inhibitors of nematode phosphoethanolamine n-methyltransferase-likeproteins can also be used to identify phosphoethanolaminen-methyltransferase-like proteins in the nematode or other organismsusing procedures known in the art, such as affinity chromatography. Forexample, a specific antibody may be linked to a resin and a nematodeextract passed over the resin, allowing any PEAMT-like proteins thatbind the antibody to bind the resin. Subsequent biochemical techniquesfamiliar to those skilled in the art can be performed to purify andidentify bound PEAMT-like proteins.

Agricultural Compositions

A compound that is identified as a phosphoethanolaminen-methyltransferase-like polypeptide inhibitor can be formulated as acomposition that is applied to plants, soil, or seeds in order to confernematode resistance. The composition can be prepared in a solution,e.g., an aqueous solution, at a concentration from about 0.005% to 10%,or about 0.01% to 1%, or about 0.1% to 0.5% by weight. The solution caninclude an organic solvent, e.g., glycerol or ethanol. The compositioncan be formulated with one or more agriculturally acceptable carriers.Agricultural carriers can include: clay, talc, bentonite, diatomaceousearth, kaolin, silica, benzene, xylene, toluene, kerosene,N-methylpyrrolidone, alcohols (methanol, ethanol, isopropanol,n-butanol, ethylene glycol, propylene glycol, and the like), and ketones(acetone, methylethyl ketone, cyclohexanone, and the like). Theformulation can optionally further include stabilizers, spreadingagents, wetting extenders, dispersing agents, sticking agents,disintegrators, and other additives, and can be prepared as a liquid, awater-soluble solid (e.g., tablet, powder or granule), or a paste.

Prior to application, the solution can be combined with another desiredcomposition such as another anthelmintic agent, germicide, fertilizer,plant growth regulator and the like. The solution may be applied to theplant tissue, for example, by spraying, e.g., with an atomizer, bydrenching, by pasting, or by manual application, e.g., with a sponge.The solution can also be distributed from an airborne source, e.g., anaircraft or other aerial object, e.g., a fixture mounted with anapparatus for spraying the solution, the fixture being of sufficientheight to distribute the solution to the desired plant tissues.Alternatively, the composition can be applied to plant tissue from avolatile or airborne source. The source is placed in the vicinity of theplant tissue and the composition is dispersed by diffusion through theatmosphere. The source and the plant tissue to be contacted can beenclosed in an incubator, growth chamber, or greenhouse, or can be insufficient proximity that they can be outdoors.

If the composition is distributed systemically thorough the plant, thecomposition can be applied to tissues other than the leaves, e.g., tothe stems or roots. Thus, the composition can be distributed byirrigation. The composition can also be injected directly into roots orstems.

A skilled artisan would be able to determine an appropriate dosage forformulation of the active ingredient of the composition. For example,the ED50 can be determined as described above from experimental data.The data can be obtained by experimentally varying the dose of theactive ingredient to identify a dosage effective for killing a nematode,while not causing toxicity in the host plant or host animal (i.e.non-nematode animal).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A purified polypeptide comprising an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO:7, whereinthe polypeptide has methyltransferase activity.
 2. A purifiedpolypeptide comprising an amino acid sequence that is at least 98%identical to the amino acid sequence of SEQ ID NO:7, wherein thepolypeptide has methyltransferase activity.
 3. A purified polypeptidecomprising the amino acid sequence of SEQ ID NO:7.